Cloning and expression of human SLAP-2: a novel SH2/ SH3 domain-containing human SLAP homologue having immune cell-specific expression

ABSTRACT

The present invention describes a newly discovered full-length polynucleotide encoding an SH2/SH3 domain-containing adapter protein, called hSLAP-2, cloned, isolated and identified. Also described are the hSLAP-2 polypeptide sequence, expression vectors, host cells, agonists, antagonists, anti-sense molecules, and antibodies related to the polynucleotide and/or polypeptide of the present invention. Methods for screening for modulators, particularly inhibitors, of the hSLAP-2 protein and use of the hSLAP-2 polynucleotide and polypeptide for therapeutics and diagnostics are described.

This application claims benefit to provisional application U.S. Ser. No.60/252,545, filed Nov. 22, 2000.

FIELD OF THE INVENTION

The invention relates to the identification and cloning of a novelfull-length human SLAP-2 gene and its encoded polypeptide product, SLAP(Src-Like Adapter Protein), which contains an SH2 (Src homology 2)domain and an SH3 (Src homology 3) domain. By homology analysis, SLAP-2is a member of the SLAP family of adapter proteins and is expressedprimarily in immune cells. The invention further relates to the use ofthe novel SLAP-2 gene and its encoded product as targets for therapeuticintervention in immunological and inflammatory disorders, autoimmunediseases, pulmonary diseases, and cancer.

BACKGROUND OF THE INVENTION

Receptor signaling pathways and intracellular signaling by receptortyrosine kinases are intimately involved in cell growth anddifferentiation. The binding of a particular growth factor or cellularligand to its receptor on a cell's plasma membrane can stimulate a widevariety of biochemical responses, including changes in ion fluxes,activation of various kinases, alteration of cell shape, transcriptionof various genes and modulation of enzymatic activities in cellularmetabolism.

Many cell receptors are tyrosine kinases whose signaling is dependentupon tyrosine phosphorylation of both the receptor and other molecules.Specific phosphorylated tyrosine residues on these receptors recruitsoluble intracellular signaling molecules to the receptor-ligand complexupon extracellular ligand stimulation, thus initiating the intracellularsignaling cascade that involves secondary signal transducer moleculesgenerated by the activated receptor. The signal can then proceed througha series of steps to the nucleus and other subcellular locations wherethe final effects of activation by the extracellular ligand areproduced. Recruitment of other molecules in the signaling pathway isoften accomplished by adapter molecules, which contain onlyprotein-protein interaction domains (e.g., SH2 and SH3 domains) and haveno associated enzymatic activity. By isolating and characterizing theadapter proteins and the molecules that interact with these adapters,important components of the signaling mechanism can be discovered,monitored and controlled.

For example, one such adapter protein is Grb2, a 24-25 kDa cytosolicadapter protein containing two SH3 domains flanking an SH2 domain, whichis known to be involved in linking many important molecules inreceptor-ligand signal transduction (E. J. Lowenstein et al., 1992,Cell, 70:431-442 and J. Downward, 1994, FEBS Letters, 338:113-117). Thecentral SH2 domain of Grb2 binds to an autophosphorylation site on thereceptor and the two flanking SH3 domains link to intracellular effectortarget molecules. An example of one such target molecule is themammalian homologue of the Drosophila ‘son of sevenless’ (SOS) protein,which is a guanine nucleotide exchange factor for ras; thus, Grb2 linksreceptors with the ras signal transduction pathway. It is now known thatthe SH3 domains also link to a number of other proteins involved in thesignaling pathway, including Vav (R. Ren et al., 1994, Genes Dev.,8:783-795; J. Wu et al., 1996, Immunity, 4:593; and L. Tuosto et al.,1996, J. Exp. Med., 184:1161); c-abl (Z. S. Ye and D. Baltimore, 1994,Proc. Natl. Acad. Sci., USA, 91:12629-12633); dynamin (I. Gout et al.,1993, Cell, 75:25-36); and SLP-76 (J. K. Jackman et al., 1995, J. Biol.Chem., 270:7029-7032). In addition, several other binding proteins havebeen noted during B- and T-cell signaling (see, e.g., K. Reif et al.,1994, J. Biol. Chem., 269:14081-14087 and D. G. Motto et al., 1994, J.Biol. Chem., 269:21608-21613).

The SLP-76 family of adapter protein molecules includes the SLP-76, BLNKand Clnk proteins (P. S. Myung et al., 2000, “Adapter proteins inlymphocyte antigen-receptor signaling”, Curr. Opin. Immunol., 12:256-266and M. Y. Cao et al., 1999, “Clnk, a novel SLP-76-related adaptermolecule expressed in cytokine-stimulated hemopoietic cells”, J. Exp.Med., 190:1527-1534). Expressed exclusively in cells of hematopoieticorigin, these adapter proteins are involved in intracellular signaltransduction. SLP-76 is an SH2/SH3 domain-containing 76 kDa leukocyteprotein that undergoes tyrosine phosphorylation following activation ofthe T-cell antigen receptor (TCR). SLP-76, upon tyrosinephosphorylation, interacts with Grb2 and phospholipase C-γ (PLC-γ), (J.K. Jackman et al., supra). The phosphorylation of SLP-76 on tyrosine isrequired for TCR-mediated cytokine secretion.

SH2 domain-containing proteins bind phosphorylated tyrosine residues andtransmit important intracellular signals in many cell types. In theimmune system, SH2 domain-containing proteins, such as SLP-76 and BLNK,play crucial roles in T-cell and B-cell activation. Therefore, SH2domain-containing proteins are likely to be important targets fortherapeutic intervention in immunological disorders, includingautoimmune disorders and inflammatory indications.

With particular regard to B-cells, cell function is dependent on theability of the membrane B-cell receptor (BCR) to bind to antigen andinduce an efficient cascade of intracellular biochemical signalingevents from the membrane to the nucleus. These events culminate in thecytosol to rearrange the morphology of the cell through cytoskeletalreorganization and in the nucleus to activate the transcription of newgenes to promote cellular proliferation and differentiation. Suchbiochemical and cellular mechanisms are required for B-cells to matureand function to produce an efficient immune response to foreignpathogens. Conversely, the abnormal activation of B-cells can lead tounregulated cellular proliferation and uncontrolled clonal expansion,resulting in B-cell tumors, lymphomas and leukemias. In addition,unregulated activation of B-cells may also contribute to a variety ofautoimmune diseases mediated by self-reactive antibodies.

In the case of T-cells, unregulated activation of the TCR can lead toaberrant T-cell growth, resulting in, for example, T-cell tumors,lymphomas, leukemias and thymomas. Thus, the ability to modulate TCR-and BCR-mediated signaling events may provide a rational approach to thetreatment of T- and B-cell mediated tumors, and the like, as well asprovide therapies for autoimmune diseases in which aberrant B-cellactivation may be the culprit for cell destruction by auto-reactiveantibodies.

Because aberrant or uncontrolled regulation of the cellular processesinvolved in cell growth can have disastrous effects, it is important toelucidate and gain control over these processes. This requiresidentifying molecules that participate in the signaling events that leadto mitogenesis and dissecting their functions and mechanisms of action.The identification of these participants is important for a wide rangeof diagnostic, therapeutic and screening applications. Morespecifically, by understanding the structure of a particular participantin a receptor ligand activation cascade, one can rationally designcompounds that affect that cascade, to either activate an otherwiseinactive pathway, or inactivate an overly active pathway.

Similarly, having identified a particular molecule in a ligand receptorcascade, situations in which that cascade is defective can also beidentified and intervention can be achieved by means of therapeuticcompounds or drugs, to prevent the development of a particularpathological state. The identification of participants in particularreceptor ligand activation cascades and intracellular signaling eventsis thus of critical importance for screening compounds that affect thesecascades and events, and for treating a variety of disorders resultingfrom anomalies in these cascades and events as therapeutic agents. Thepresent invention meets these and several additional needs.

Also, the discovery of human SLAP-2, a new member of the SLAP family ofadapter proteins, and the polynucleotide encoding this protein, providesthe art with new compositions and methods of use and treatment for thediagnosis, screening, monitoring, therapy, and prevention of immunesystem related conditions or diseases, particularly those involvingT-cell and B-cell neoplasms; inflammation disorders, diseases andconditions, rheumatoid arthritis, osteoarthritis, psoriasis, rhinitis,inflammatory bowel disease (Crohn's and ulcerative colitis), allergies,particularly those involving hyperactivity of B-cells and T-cells, orother immune cells, such as mast cells or eosinophils; autoimmunediseases such as systemic lupus erythematosus and multiple sclerosis;pulmonary diseases including asthma, acute respiratory distresssyndrome, and chronic obstructive pulmonary disorder; tissue/organrejection; and cancer.

SUMMARY OF THE INVENTION

The present invention provides a newly discovered full-length humanSH2-/SH3-domain containing gene and its encoded product, called hSLAP-2(Human Src-Like Adapter Protein-2), which has homology to hSLAP (hSLAP)and mSLAP (mouse SLAP).

It is an object of the present invention to provide an isolatedfull-length hSLAP-2 polynucleotide as depicted in SEQ ID NO:1. Thepresent invention also provides a polynucleotide sequence comprising thecomplement of SEQ ID NO:1, or variants thereof. In addition, the presentinvention features polynucleotide sequences which hybridize undermoderate or high stringency conditions to the polynucleotide sequence ofSEQ ID NO:1.

It is another object of the present invention to provide the humanhSLAP-2 polypeptide, encoded by the polynucleotide of SEQ ID NO:1 andhaving the amino acid sequence of SEQ ID NO:2, or a functional orbiologically active portion thereof. In accordance with the presentinvention, an isolated, substantially purified full-length human SLAP-2protein is provided.

It is a further object of the present invention to provide compositionscomprising the human SLAP-2 polynucleotide sequence, or a fragmentthereof, or the encoded hSLAP-2 polypeptide, or a fragment or portionthereof. Also provided by the present invention are pharmaceuticalcompositions comprising at least one hSLAP-2 polypeptide, or afunctional portion thereof, wherein the compositions further comprise apharmaceutically acceptable carrier, excipient, or diluent.

It is a further object of the invention to provide an anti-sense of thehuman SLAP-2 nucleic acid sequence, as well as oligonucleotides,fragments, or portions of the hSLAP-2 nucleic acid molecule oranti-sense molecule. Also provided are expression vectors and host cellscomprising polynucleotides that encode the human SLAP-2 polypeptide, orportions or fragments thereof.

It is an object of the present invention to provide methods forproducing a polypeptide comprising the amino acid sequence depicted inSEQ ID NO:2, or a fragment thereof, comprising the steps of a)cultivating a host cell containing an expression vector containing atleast a functional fragment of the polynucleotide sequence encoding thehuman SLAP-2 polypeptide according to this invention under conditionssuitable for the expression of the polynucleotide; and b) recovering thepolypeptide from the host cell.

It is a further object of the present invention to provide antibodies,and binding fragments thereof, which bind specifically to the hSLAP-2polypeptide, or an epitope thereof, for use as therapeutics anddiagnostic agents.

It is an object of the present invention to provide methods forscreening for agents or molecules which bind to and/or modulate humanSLAP-2 polypeptide, e.g., inhibitors, other intracellular signalingmolecules and antagonists, as well as the modulators, particularly,inhibitors and antagonists, particularly those that are obtained fromthe screening methods described. Also provided are methods to screen forinhibitors of the interaction, e.g., a binding interaction, of thehSLAP-2 protein with other signaling proteins, particularly those havingSH2 and SH3 interaction domains.

It is also an object of the present invention to provide a substantiallypurified antagonist or inhibitor of the polypeptide of SEQ ID NO:2. Inthis regard, and by way of example, a purified antibody that binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:2 isprovided.

It is a further object of the present invention to provide hSLAP-2nucleic acid sequences, polypeptide, peptides and antibodies for use inthe diagnosis and/or screening of disorders or diseases associated withexpression of the polynucleotide and its encoded polypeptide asdescribed herein.

It is an object of the present invention to provide kits for screeningand diagnosis of disorders associated with aberrant or uncontrolledcellular development and with the expression of the hSLAP-2polynucleotide and its encoded polypeptide as described herein.

It is an object of the present invention to further provide methods forthe treatment or prevention of immune cell disorders or diseases, e.g.,B- or T-cell tumors, lymphomas, leukemias, autoimmune diseases, orinflammation, involving administering to an individual in need oftreatment or prevention an effective amount of a purified antagonist oragonist of the hSLAP-2 polypeptide. It is an object of the presentinvention to provide a method for detecting a polynucleotide thatencodes the hSLAP-2 polypeptide in a biological sample comprising thesteps of: a) hybridizing the complement of the polynucleotide sequenceencoding SEQ ID NO:2 to a nucleic acid material of a biological sample,thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding the hSLAP-2 polypeptidein the biological sample. The nucleic acid material may be furtheramplified by the polymerase chain reaction prior to hybridization.

Further objects, features and advantages of the present invention willbe better understood upon a reading of the detailed description of theinvention when considered in connection with the accompanyingfigures/drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the full-length polynucleotide sequence of human SLAP-2cDNA of the present invention (SEQ ID NO:1; clone 13).

FIG. 2 shows the amino acid sequence comprising the hSLAP-2 polypeptide(SEQ ID NO:2).

FIGS. 3A-3B show the nucleic acid sequence of human SLAP-2 cDNA (SEQ IDNO:1), and the deduced, encoded amino acid sequence of the human SLAP-2gene product (SEQ ID NO:2). The SH3 domain is boxed. The SH2 domain isunderlined. Putative tyrosine phosphorylation sites are in boldfacetype.

FIG. 4 shows the homology between human SLAP (hSLAP), murine SLAP(mSLAP), and human SLAP-2 (hSLAP-2). Results are shown in bothpercentages of similarity and identity at the amino acid level.

FIG. 5 presents an alignment of hSLAP-2 (SEQ ID NO:2) and hSLAP (SEQ IDNO:6) amino acid sequences. Lines between resides indicate identity,double dots indicate conservative differences, and single dots indicatenon-conservative differences. Individual dots above the residuesdemarcate every 10^(th) amino acid.

FIG. 6 shows the alignment of hSLAP (SEQ ID NO:6) and mSLAP (SEQ IDNO:7) amino acid sequences. Lines between residues indicate identity,double dots indicate conservative differences, and single dots indicatenon-conservative differences. Individual dots above the residuesdemarcate every 10^(th) amino acid.

FIG. 7 shows the alignment of hSLAP-2 (SEQ ID NO:2) and mSLAP (SEQ IDNO:7) amino acid sequences. Lines between residues indicate identity,double dots indicate conservative differences, and single dots indicatenon-conservative differences. Individual dots above the residuesdemarcate every 10^(th) amino acid.

FIG. 8 depicts an electronic Northern analysis of the tissue expressionof hSLAP-2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel isolated polynucleotide (SEQ IDNO:1) encoding the full-length hSLAP-2 polypeptide (SEQ ID NO:2), aprotein having similarity at the amino acid level to otherSH2/SH3-domain containing adapter proteins which function in thereceptor-ligand signal transduction pathway in cells of thehematopoietic lineage.

The following definitions are provided to more fully describe thepresent invention in its various aspects. The definitions are intendedto be useful for guidance and elucidation, and are not intended to limitthe disclosed invention and its embodiments.

Definitions

The “hSLAP-2 polypeptide” (or protein) refers to the amino acid sequenceof substantially purified hSLAP-2, which, although isolated from a humancDNA library source according to the present invention, may be obtainedfrom any species, preferably mammalian, including mouse, rat, non-humanprimates, and more preferably, human; and from a variety of sources,including natural, synthetic, semi-synthetic, or recombinant. Functionalfragments of the hSLAP-2 polypeptide are also embraced by the presentinvention.

An “agonist” refers to a molecule which, when bound to the hSLAP-2polypeptide, or a functional fragment thereof, increases or prolongs theduration of the effect of the hSLAP-2 polypeptide. Agonists may includeproteins, nucleic acids, carbohydrates, or any other molecules that bindto and modulate the effect of hSLAP-2 polypeptide. An “antagonist”(e.g., inhibitor) refers to a molecule which, when bound to the hSLAP-2polypeptide, or a functional fragment thereof, decreases or eliminatesthe amount or duration of the biological or immunological activity ofhSLAP-2 polypeptide. Antagonists may include proteins, nucleic acids,carbohydrates, antibodies, or any other molecules that decrease, reduceor eliminate the effect of the hSLAP-2 polypeptide.

“Nucleic acid sequence”, as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or anti-sense strand. By way ofnonlimiting example, fragments include nucleic acid sequences that aregreater than 20-60 nucleotides in length, and preferably includefragments that are at least 70-100 nucleotides, or which are at least1000 nucleotides or greater in length. Nucleic acids for use as probesor primers may differ in length as described herein.

Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.Amino acid sequence fragments are typically from about 4 or 5 to about35, preferably from about 5 to about 15 or 20 amino acids in length and,optimally, retain the biological activity or function of the hSLAP-2polypeptide.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. In addition,the terms “hSLAP-2 polypeptide” and “hSLAP-2 protein” are frequentlyused interchangeably herein to refer to the encoded product of thehSLAP-2 nucleic acid sequence of the present invention.

A “variant” of the hSLAP-2 polypeptide can refer to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing functional biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

An “allele” or “allelic sequence” is an alternative form of the hSLAP-2nucleic acid sequence. Alleles may result from at least one mutation inthe nucleic acid sequence and may yield altered mRNAs or polypeptideswhose structure or function may or may not be altered. Any given gene,whether natural or recombinant, may have none, one, or many allelicforms. Common mutational changes, which give rise to alleles, aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Altered nucleic acid sequences encoding the hSLAP-2 polypeptide includenucleic acid sequences containing deletions, insertions and/orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent hSLAP-2 polypeptide.Altered nucleic acid sequences may further include polymorphisms of thepolynucleotide encoding the hSLAP-2 polypeptide; such polymorphisms mayor may not be readily detectable using a particular oligonucleotideprobe. The encoded protein may also contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in a functionally equivalent hSLAP-2 protein of the presentinvention. Deliberate amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological activity or function of hSLAP-2 protein is retained.For example, negatively charged amino acids may include aspartic acidand glutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide (“oligo”) linked viaan amide bond, similar to the peptide backbone of amino acid residues.PNAs typically comprise oligos of at least 5 nucleotides linked viaamide bonds. PNAs may or may not terminate in positively charged aminoacid residues to enhance binding affinities to DNA. Such amino acidsinclude, for example, lysine and arginine, among others. These smallmolecules stop transcript elongation by binding to their complementarystrand of nucleic acid (P. E. Nielsen et al., 1993, Anticancer DrugDes., 8:53-63). PNA may be pegylated to extend their lifespan in thecell where they preferentially bind to complementary single stranded DNAand RNA.

“Oligonucleotides” or “oligomers” refer to a nucleic acid sequence,preferably comprising contiguous nucleotides, of at least about 6nucleotides to about 60 nucleotides, preferably at least about 8 to 10nucleotides in length, more preferably at least about 12 nucleotides inlength, e.g., about 15 to 35 nucleotides, or about 15 to 25 nucleotides,or about 20 to 35 nucleotides, which can be typically used, for example,as probes or primers, in PCR amplification assays, hybridization assays,or in microarrays. It will be understood that the term oligonucleotideis substantially equivalent to the terms primer, probe, or amplimer, ascommonly defined in the art. It will also be appreciated by thoseskilled in the pertinent art that a longer oligonucleotide probe, ormixtures of probes, e.g., degenerate probes, can be used to detectlonger, or more complex, nucleic acid sequences, for example, genomicDNA. In such cases, the probe may comprise at least 20-200 nucleotides,preferably, at least 30-100 nucleotides, and more preferably, 50-100nucleotides.

“Amplification” refers to the production of additional copies of anucleic acid sequence and is generally carried out using polymerasechain reaction (PCR) technologies, which are well known and practiced inthe art (see, D. W. Dieffenbach and G. S. Dveksler, 1995, PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

“Microarray” is an array of distinct polynucleotides or oligonucleotidessynthesized on a substrate, such as paper, nylon, or other type ofmembrane; filter; chip; glass slide; or any other type of suitable solidsupport.

The term “antisense” refers to nucleotide sequences, and compositionscontaining nucleic acid sequences, which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense (i.e., complementary) nucleic acid molecules include PNA andmay be produced by any method, including synthesis or transcription.Once introduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes, which blockeither transcription or translation. The designation “negative” issometimes used in reference to the antisense strand, and “positive” issometimes used in reference to the sense strand.

The term “consensus” refers to the sequence that reflects the mostcommon choice of base or amino acid at each position among a series ofrelated DNA, RNA or protein sequences. Areas of particularly goodagreement often represent conserved functional domains.

A “deletion” refers to a change in either nucleotide or amino acidsequence and results in the absence of one or more nucleotides or aminoacid residues. By contrast, an insertion (also termed “addition”) refersto a change in a nucleotide or amino acid sequence that results in theaddition of one or more nucleotides or amino acid residues, as comparedwith the naturally occurring molecule. A “substitution” refers to thereplacement of one or more nucleotides or amino acids by differentnucleotides or amino acids.

A “derivative nucleic acid molecule” refers to the chemical modificationof a nucleic acid encoding, or complementary to, the encoded hSLAP-2polypeptide. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide, which retains the essential biological and/orfunctional characteristics of the natural molecule. A derivativepolypeptide is one which is modified by glycosylation, pegylation, orany similar process that retains the biological and/or functional orimmunological activity of the polypeptide from which it is derived.

The term “biologically active”, i.e., functional, refers to a protein orpolypeptide or peptide fragment thereof having structural, regulatory,or biochemical functions of a naturally occurring molecule. Likewise,“immunologically active” refers to the capability of the natural,recombinant, or synthetic hSLAP-2, or any oligopeptide thereof, toinduce a specific immune response in appropriate animals or cells, forexample, to generate antibodies, and to bind with specific antibodies.

The term “hybridization” refers to any process by which a strand ofnucleic acid binds with a complementary strand through base pairing.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary G and C bases and between complementary A and Tbases. The hydrogen bonds may be further stabilized by base stackinginteractions. The two complementary nucleic acid sequences hydrogen bondin an anti-parallel configuration. A hybridization complex may be formedin solution (e.g., C_(o)t or R_(o)t analysis), or between one nucleicacid sequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., membranes, filters, chips, pins,or glass slides, or any other appropriate substrate to which cells ortheir nucleic acids have been affixed).

The terms “stringency” or “stringent conditions” refer to the conditionsfor hybridization as defined by nucleic acid composition, salt andtemperature. These conditions are well known in the art and may bealtered to identify and/or detect identical or related polynucleotidesequences in a sample. A variety of equivalent conditions comprisingeither low, moderate, or high stringency depend on factors such as thelength and nature of the sequence (DNA, RNA, base composition), reactionmilieu (in solution or immobilized on a solid substrate), nature of thetarget nucleic acid (DNA, RNA, base composition), concentration of saltsand the presence or absence of other reaction components (e.g.,formamide, dextran sulfate and/or polyethylene glycol) and reactiontemperature (within a range of from about 5° C. below the meltingtemperature of the probe to about 20° C. to 25° C. below the meltingtemperature). One or more factors may be varied to generate conditions,either low or high stringency, that is different from but equivalent tothe aforementioned conditions.

As will be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences. As will be further appreciated by theskilled practitioner, the melting temperature, Tm, can be approximatedby the formulas as known in the art, depending on a number ofparameters, such as the length of the hybrid or probe in number ofnucleotides, or hybridization buffer ingredients and conditions (see,for example, T. Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982 and J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989; Current Protocols inMolecular Biology, Eds. F. M. Ausubel et al., Vol. 1, “Preparation andAnalysis of DNA”, John Wiley and Sons, Inc., 1994-1995, Suppls. 26, 29,35 and 42; pp. 2.10.7-2.10.16; G. M. Wahl and S. L. Berger (1987;Methods Enzymol. 152:399-407); and A. R. Kimmel, 1987; Methods ofEnzymol. 152:507-511). As a general guide, Tm decreases approximately 1°C.-1.5° C. with every 1% decrease in sequence homology. Also, ingeneral, the stability of a hybrid is a function of sodium ionconcentration and temperature. Typically, the hybridization reaction isinitially performed under conditions of low stringency, followed bywashes of varying, but higher stringency. Reference to hybridizationstringency, e.g., high, moderate, or low stringency, typically relatesto such washing conditions.

Thus, by way of non-limiting example, “high stringency” refers toconditions that permit hybridization of those nucleic acid sequencesthat form stable hybrids in 0.018 M NaCl at about 65° C. (i.e., if ahybrid is not stable in 0.018 M NaCl at about 65° C., it will not bestable under high stringency conditions). High stringency conditions canbe provided, for instance, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE (saline sodium phosphate EDTA) (1×SSPEbuffer comprises 0.15 M NaCl, 10 mM Na₂HPO₄, 1 mM EDTA), (or 1×SSCbuffer containing 150 mM NaCl, 15 mM Na₃ citrate•2H₂O, pH 7.0), 0.2% SDSat about 42° C., followed by washing in 1×SSPE (or saline sodiumcitrate, SSC) and 0.1% SDS at a temperature of at least about 42° C.,preferably about 55° C., more preferably about 65° C.

“Moderate stringency” refers, by nonlimiting example, to conditions thatpermit hybridization in 50% formamide, 5× Denhardt's solution, 5×SSPE(or SSC), 0.2% SDS at 42° C. (to about 50° C.), followed by washing in0.2×SSPE (or SSC) and 0.2% SDS at a temperature of at least about 42°C., preferably about 55° C., more preferably about 65° C.

“Low stringency” refers, by non-limiting example, to conditions thatpermit hybridization in 10% formamide, 5× Denhardt's solution, 6×SSPE(or SSC), 0.2% SDS at 42° C., followed by washing in 1×SSPE (or SSC) and0.2% SDS at a temperature of about 45° C., preferably about 50° C.

For additional stringency conditions, see T. Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982). It is to be understood that the low, moderate andhigh stringency hybridization/washing conditions may be varied using avariety of ingredients, buffers and temperatures well known to andpracticed by the skilled practitioner.

The terms “complementary” or “complementarity” refer to the naturalbinding of polynucleotides under permissive salt and temperatureconditions by base pairing. For example, the sequence “A-G-T” binds tothe complementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, in which only some of thenucleic acids bind, or it may be complete when total complementarityexists between single-stranded molecules. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, which depend uponbinding between nucleic acids strands, as well as in the design and useof PNA molecules.

The term “homology” refers to a degree of complementarity. There may bepartial sequence homology or complete homology, wherein “completehomology” is equivalent to identity, e.g., 100% identity. A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to usingthe functional term “substantially homologous”. The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (e.g., Southern orNorthern blot, solution hybridization, and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (i.e., the hybridization) of acompletely homologous sequence or probe to the target sequence underconditions of low stringency. Nonetheless, conditions of low stringencydo not permit non-specific binding; low stringency conditions requirethat the binding of two sequences to one another be a specific (i.e.,selective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% identity). In theabsence of non-specific binding, the probe will not hybridize to thesecond non-complementary target sequence.

Those having skill in the art will know how to determine percentidentity between/among sequences using, for example, algorithms such asthose based on the CLUSTALW computer program (J. D. Thompson et al.,1994, Nucleic Acids Research, 2(22):4673-4680), or FASTDB, (Brutlag etal., 1990, Comp. App. Biosci., 6:237-245), as known in the art. Althoughthe FASTDB algorithm typically does not consider internal non-matchingdeletions or additions in sequences, i.e., gaps, in its calculation,this can be corrected manually to avoid an overestimation of the %identity. CLUSTALW, however, does take sequence gaps into account in itsidentity calculations.

A “composition comprising a given polynucleotide sequence” refersbroadly to any composition containing the given polynucleotide sequence.The composition may comprise a dry formulation or an aqueous solution.Compositions comprising the polynucleotide sequence (SEQ ID NO:1)encoding hSLAP-2 polypeptide (SEQ ID NO:2), or fragments thereof, may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be in association with a stabilizing agentsuch as a carbohydrate. In hybridizations, the probe may be employed inan aqueous solution containing salts (e.g., NaCl), detergents orsurfactants (e.g., SDS), and other components (e.g., Denhardt'ssolution, dry milk, salmon sperm DNA, and the like).

The term “substantially purified” refers to nucleic acid sequences oramino acid sequences that are removed from their natural environment,i.e., isolated or separated by a variety of means, and are at least 60%free, preferably 75% to 85% free, and most preferably 90% or greaterfree from other components with which they are naturally associated.

The term “sample”, or “biological sample”, is meant to be interpreted inits broadest sense. A biological sample suspected of containing nucleicacid encoding the hSLAP-2 protein, or fragments thereof, or the hSLAP-2protein itself, may comprise a body fluid, an extract from cells ortissue, chromosomes isolated from a cell (e.g., a spread of metaphasechromosomes), organelle, or membrane isolated from a cell, a cell,nucleic acid such as genomic DNA (in solution or bound to a solidsupport such as for Southern analysis), RNA (in solution or bound to asolid support such as for Northern analysis), cDNA (in solution or boundto a solid support), a tissue, a tissue print and the like.

“Transformation” refers to a process by which exogenous DNA enters andchanges a recipient cell. It may occur under natural or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method isselected based on the type of host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation, heatshock, lipofection, and partial bombardment. Such “transformed” cellsinclude stably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. Transformed cells also include those cells whichtransiently express the inserted DNA or RNA for limited periods of time.

The term “mimetic” refers to a molecule, the structure of which isdeveloped from knowledge of the structure of the hSLAP-2 protein, orportions thereof, and as such, is able to affect some or all of theactions of the hSLAP-2 protein.

The term “portion” with regard to a protein (as in “a portion of a givenprotein”) refers to fragments or segments, for example, peptides, ofthat protein. The fragments may range in size from four or five aminoacid residues to the entire amino acid sequence minus one amino acid.Thus, a protein “comprising at least a portion of the amino acidsequence of SEQ ID NO: 2” encompasses the full-length human hSLAP-2polypeptide, and fragments thereof.

The term “antibody” refers to intact molecules as well as fragmentsthereof, such as Fab, F(ab′)₂, Fv, which are capable of binding anepitopic or antigenic determinant. Antibodies that bind to hSLAP-2polypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest or prepared recombinantly for useas the immunizing antigen. The polypeptide or oligopeptide used toimmunize an animal can be derived from the transition of RNA orsynthesized chemically, and can be conjugated to a carrier protein, ifdesired. Commonly used carriers that are chemically coupled to peptidesinclude bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g, a mouse, a rat, or a rabbit).

The term “humanized” antibody refers to antibody molecules in whichamino acids have been replaced in the non-antigen binding regions inorder to more closely resemble a human antibody, while still retainingthe original binding capability, e.g., as described in U.S. Pat. No.5,585,089 to C. L. Queen et al.

The term “antigenic determinant” refers to that portion of a moleculethat makes contact with a particular antibody (i.e., an epitope). When aprotein or fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to a given region or three-dimensional structureon the protein; these regions or structures are referred to as antigenicdeterminants. An antigenic determinant may compete with the intactantigen (i.e., the immunogen used to elicit the immune response) forbinding to an antibody.

The terms “specific binding” or “specifically binding” refer to theinteraction between a protein or peptide and a binding molecule, such asan agonist, an antagonist, or an antibody. The interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope, or a structural determinant) of the protein thatis recognized by the binding molecule. For example, if an antibody isspecific for epitope “A”, the presence of a protein containing epitope A(or free, unlabeled A) in a reaction containing labeled “A” and theantibody will reduce the amount of labeled A bound to the antibody. Inaddition, the hSLAP-2 protein of the present invention contains anSH2/SH3 domain that serves as an interacting region of hSLAP-2 withother cellular proteins, putative tyrosine residues that may becomephosphorylated and could bind to SH2 domains on other cellular proteinsand an SH3 binding motif that may serve as a binding domain for othercellular proteins having an SH3 domain. (FIGS. 3A-3B).

The term “correlates with expression of a polynucleotide” indicates thatthe detection of the presence of ribonucleic acid that is similar to SEQID NO 1 by Northern analysis is indicative of the presence of mRNAencoding the hSLAP-2 polypeptide in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

An “alteration in the polynucleotide of SEQ ID NO:1” comprises anyalteration in the sequence of the polynucleotides encoding the hSLAP-2polypeptide, including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes the hSLAP-2 polypeptide (e.g., by alterations in thepattern of restriction fragment length polymorphisms capable ofhybridizing to SEQ ID NO:1), the inability of a selected fragment of SEQID NO:1 to hybridize to a sample of genomic DNA (e.g., usingallele-specific oligonucleotide probes), and improper or unexpectedhybridization, such as hybridization to a locus other than the normalchromosomal locus for the polynucleotide sequence encoding the hSLAP-2polypeptide (e.g., using fluorescent in situ hybridization (FISH) tometaphase chromosome spreads).

DESCRIPTION OF THE PRESENT INVENTION

The present invention is based on the discovery of a novel full-lengthhuman Src homology 2-/Src homology 3-(SH2/SH3) domain-containing geneand its encoded protein, called hSLAP-2, which was determined byhomology analysis to be a member of the SLAP family of adapter proteins.The gene and encoded product according to the present invention arecalled hSLAP-2 (human Src-Like Adapter Protein-2) due to its similaritywith both human SLAP (hSLAP) and mouse SLAP (mSLAP) sequences. The SLAPproteins have been shown to be negative regulators of intracellularsignal transduction in several cell types, including T-cells (see:Roche, S. et al., (1998) Src-like adaptor protein (Slap) is a negativeregulator of mitogenesis. Curr. Biol. 8:975-978; Tang, J. et al., (1999)SLAP, a dimeric adapter protein, plays a functional role in T cellreceptor signaling. Proc. Natl. Acad. Sci. USA 96:9775-9780; andSosinowski, T. et al., (2000) Src-like adaptor protein (SLAP) is anegative regulator of T cell receptor signaling. J. Exp. Med.191:463-474).

hSLAP-2 Polynucleotides and Polypeptides

The present invention encompasses the nucleic acid sequence (SEQ IDNO:1) encoding the full-length hSLAP-2 polypeptide (SEQ ID NO:2) and theuse of compositions comprising the hSLAP-2 polynucleotide or polypeptidein methods for screening for antagonists or inhibitors of theinteraction of hSLAP-2 with cellular signaling components. Alsoencompassed by the invention is the use of the hSLAP-2 nucleic acidsequence and the hSLAP-2 polypeptide in methods for diagnosing, treatingor preventing disorders or diseases associated with aberrant oruncontrolled cellular signal transduction or with hyperactive cells,particularly in cells of immunological origin, including B- andT-lymphocytes, monocytes, mast cells and the like. Immunological orinflammatory disorders such as rheumatoid arthritis, rejection of organor tissue transplants, inflammatory bowel disorders; autoimmune diseasessuch as systemic lupus erythematosus and multiple sclerosis; pulmonarydiseases including asthma and chronic obstructive pulmonary disorder;and cancer, are particular targets for treatment by the presentinvention, including inhibitors of hSLAP-2 polynucleotide andpolypeptide function. In addition, the hSLAP-2 gene and polypeptide areuseful for determining those cellular signaling molecules whichassociate with hSLAP-2 and which provide critical signals for cellactivation, preferably, T-cell activation.

According to the present invention, nucleic acids encoding human hSLAP-2protein was first identified as a PCR product in a human leukocyte cDNAlibrary, as described in Example 1. In addition, upon screening a humanleukocyte cDNA library with the GeneTrapper™ (Life Technologies, Inc.;Gaithersburg, Md.) primer, positive clones containing the novel sequencewere also identified. End-sequencing analysis revealed that one of theclones (clone #13) contains the full-length coding region of the hSLAP-2gene, as described in Example 1.

In one of its embodiments, the present invention encompasses apolypeptide comprising the amino acid sequence of SEQ ID NO:2 as shownin FIG. 2. The human hSLAP-2 polypeptide is 262 amino acids in lengthand shares amino acid sequence similarity to the SLAP family members,hSLAP and mSLAP as presented in FIG. 4.

The table of FIG. 4 shows the percent similarity/identity at the aminoacid level between human SLAP and mouse SLAP protein; between human SLAPand human SLAP-2; and between mouse SLAP and human SLAP-2. Based on thecomparative data, human SLAP-2 is determined to be a novel sequence fromthis family of adapter proteins.

A mouse SLAP protein was reported by Pandey et al. (Pandey, a. et al.(1995) Characterization of a novel Src-like adapter protein thatassociates with the Eck receptor tyrosine kinase. J. Biol. Chem. 270:19201-19204), followed by the identification of a human counterpart SLAPgene (Angrist, M. et al. (1995) Chromosomal localization of the mouseSrc-like adapter protein (Slap) gene and its putative human homologueSLA. Genomics 30: 623-625; Meijerink, P. H. et al. (1998). The gene forthe human Src-like adapter protein (hSLAP) is located within the 64-kbintron of the thyroglobulin gene. Eur. J. Biochem. 254: 297-303). MouseSLAP is predominantly expressed in lymphoid cells (Sosinowski, T. et al.(2000) Src-like adapter protein (SLAP) is a negative regulator of T cellreceptor signaling. J. Exptl. Med. 191: 463-474), and hSLAP-2 has asimilar expression pattern, as seen in FIG. 8.

Variants of the hSLAP-2 polypeptide are also encompassed by the presentinvention. A preferred hSLAP-2 variant has at least 75 to 80%, morepreferably at least 85 to 90%, and even more preferably at least 90%amino acid sequence identity to the amino acid sequence (SEQ ID NO:2)disclosed herein, and which retains at least one biological,immunological, or other functional characteristic or activity of thehSLAP-2 polypeptide. Most preferred is a variant having at least 95%amino acid sequence identity to the amino acid sequence set forth in SEQID NO:2. An amino acid sequence variant of the hSLAP-2 protein can becategorized into one or more of three classes: substitutional,insertional, or deletional variants. Such variants are typicallyprepared by site-specific mutagenesis of nucleotides in the DNA encodingthe hSLAP-2 protein, using cassette or PCR mutagenesis, or othertechniques that are well known and practiced in the art, to produce DNAencoding the variant. Thereafter, the DNA is expressed in recombinantcell culture as described herein. Variant hSLAP-2 protein fragmentshaving up to about 100-150 residues may be prepared by in vitrosynthesis using conventional techniques.

Amino acid sequence variants are characterized by the predeterminednature of the variation, a feature that sets them apart from naturallyoccurring allelic or interspecies variations of the hSLAP-2 proteinamino acid sequence. The variants typically exhibit the same qualitativebiological activity as that of the naturally occurring analogue,although variants can also be selected having modified characteristics.While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be performed at thetarget codon or region, and the expressed hSLAP-2 variants screened forthe optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is accomplished using assays ofhSLAP-2 protein activities, for example, for binding domain mutations,competitive binding studies may be carried out.

Amino acid substitutions are typically of single residues; insertionsusually are on the order of from one to twenty amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout one to about 20 residues, although in some cases, deletions may bemuch larger. For example, preferred deletion variants include thedeletion of one or more of the characteristic domains, i.e., theproline-rich region, or the SH2/SH3 domain.

Substitutions, deletions, insertions, or any combination thereof, may beused to arrive at a final hSLAP-2 derivative. Generally, these changesaffect only a few amino acids to minimize the alteration of themolecule. However, larger changes may be tolerated in certaincircumstances. When small alterations in the characteristics of thehSLAP-2 protein are desired or warranted, substitutions are generallymade in accordance with the following table: TABLE 1 Exemplary OriginalResidue Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser GlnAsn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln,Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, PheVal Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTable 1. For example, substitutions may be made which more significantlyaffect the structure of the polypeptide backbone in the area of thealteration, for example, the alpha-helical, or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which generally are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) a residue that does not have a side chain, e.g., glycine.

While hSLAP-2 variants ordinarily exhibit the same qualitativebiological activity or function, and elicit the same immune response, asthe naturally occurring analogue, the variants are also selected tomodify the characteristics of the hSLAP-2 protein as needed.Alternatively, the variant may be designed such that the biologicalactivity of the hSLAP-2 protein is altered. For example, any or all ofthe domains may be altered, i.e., the SH2 and/or SH3 regions, and/or theamino- and carboxy-terminal regions outside of the SH2 and SH3 domains.For example, one or more of the tyrosine phosphorylation sites may bealtered.

In another embodiment, the present invention encompasses polynucleotideswhich encode the hSLAP-2 polypeptide. Accordingly, any nucleic acidsequence which encodes the amino acid sequence of the hSLAP-2polypeptide can be used to produce recombinant molecules that expresshSLAP-2 protein. In a particular embodiment, the present inventionencompasses the hSLAP-2 polynucleotide comprising the nucleic acidsequence of SEQ ID NO:1 and as shown in FIG. 1. More particularly, thepresent invention provides the cloned full-length hSLAP-2 cDNA,deposited at the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 on ______ and under ATCCAccession No. ______ according to the terms of the Budapest Treaty.

As will be appreciated by the skilled practitioner in the art, thedegeneracy of the genetic code results in the production of numerousnucleotide sequences encoding the hSLAP-2 polypeptide of the presentinvention. Some of the sequences bear minimal homology to the nucleotidesequences of any known and naturally occurring gene. Accordingly, thepresent invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring hSLAP-2, and all such variations are tobe considered as being specifically disclosed.

Although nucleotide sequences which encode the hSLAP-2 polypeptide andits variants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring hSLAP-2 polypeptide underappropriately selected conditions of stringency, it may be advantageousto produce nucleotide sequences encoding the hSLAP-2 polypeptide, or itsderivatives, which possess a substantially different codon usage. Codonsmay be selected to increase the rate at which expression of thepeptide/polypeptide occurs in a particular prokaryotic or eukaryotichost in accordance with the frequency with which particular codons areutilized by the host, for example, in plant cells or yeast cells oramphibian cells. Other reasons for substantially altering the nucleotidesequence encoding the hSLAP-2 polypeptide, and its derivatives, withoutaltering the encoded amino acid sequences include the production of mRNAtranscripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The present invention also encompasses production of DNA sequences, orportions thereof, which encode the hSLAP-2 polypeptide, and itsderivatives, entirely by synthetic chemistry. After production, thesynthetic sequence may be inserted into any of the many availableexpression vectors and cell systems using reagents that are well knownand practiced by those in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding hSLAP-2polypeptide, or any fragment thereof.

Also encompassed by the present invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequence ofhSLAP-2, such as that shown in SEQ ID NO:1, under various conditions ofstringency. Hybridization conditions are typically based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe (see, G.M. Wahl and S. L. Berger, 1987; Methods Enzymol., 152:399-407 and A. R.Kimmel, 1987; Methods of Enzymol., 152:507-511), and may be used at adefined stringency. For example, included in the present invention aresequences capable of hybridizing under moderately stringent conditionsto the hSLAP-2 nucleic acid sequence of SEQ ID NO:1 and other sequenceswhich are degenerate to those which encode the hSLAP-2 polypeptide(e.g., as a non-limiting example: pre-washing solution of 2×SSC, 0.5%SDS, 1.0 mM EDTA, pH 8.0, and hybridization conditions of 50° C., 5×SSC,overnight).

In another embodiment of the present invention, polynucleotide sequencesor fragments (peptides) thereof which encode the hSLAP-2 polypeptide maybe used in recombinant DNA molecules to direct the expression of thehSLAP-2 polypeptide product, or fragments or functional equivalentsthereof, in appropriate host cells. Because of the inherent degeneracyof the genetic code, other DNA sequences, which encode substantially thesame or a functionally equivalent amino acid sequence, may be producedand these sequences may be used to express hSLAP-2 protein.

As will be appreciated by those having skill in the art, it may beadvantageous to produce hSLAP-2 polypeptide-encoding nucleotidesequences possessing non-naturally occurring codons. For example, codonspreferred by a particular prokaryotic or eukaryotic host can be selectedto increase the rate of protein expression or to produce a recombinantRNA transcript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequence of the present invention can be engineered usingmethods generally known in the art in order to alter hSLAP-2polypeptide-encoding sequences for a variety of reasons, including, butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and the like.

In another embodiment of the present invention, natural, modified, orrecombinant nucleic acid sequences, or a fragment thereof, encodinghSLAP-2 polypeptide may be ligated to a heterologous sequence to encodea fusion protein. For example, for screening peptide libraries forinhibitors or modulators of hSLAP-2 activity or binding, it may beuseful to encode a chimeric hSLAP-2 protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the hSLAP-2 protein-encodingsequence and the heterologous protein sequence, so that the hSLAP-2protein may be cleaved and purified away from the heterologous moiety.

In another embodiment, sequences encoding the hSLAP-2 polypeptide may besynthesized in whole, or in part, using chemical methods well known inthe art (see, for example, M. H. Caruthers et al., 1980, Nucl. AcidsRes. Symp. Ser., 215-223 and Horn, T. et al., 1980, Nucl. Acids Res.Symp. Ser., 225-232). Alternatively, the protein itself may be producedusing chemical methods to synthesize the amino acid sequence of thehSLAP-2 polypeptide, or a fragment or portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(J. Y. Roberge et al., 1995, Science, 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431 A PeptideSynthesizer (PE Biosystems).

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., T. Creighton,1983, Proteins, Structures and Molecular Principles, WH Freeman and Co.,New York, N.Y.), by reversed-phase high performance liquidchromatography, or other purification methods as are known in the art.The composition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure;Creighton, supra). In addition, the amino acid sequence of the hSLAP-2polypeptide or any portion thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

Expression of Human hSLAP-2 Protein

To express a biologically active/functional hSLAP-2 polypeptide orpeptide, the nucleotide sequences encoding the hSLAP-2 polypeptide, orfunctional equivalents, may be inserted into an appropriate expressionvector, i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence. Methodswhich are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding the hSLAP-2polypeptide and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in J. Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F.M. Ausubel et al., 1989, Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding the hSLAP-2 polypeptide. Such expressionvector/host systems include, but are not limited to, microorganisms suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast or fungi transformed with yeast orfungal expression vectors; insect cell systems infected with virusexpression vectors (e.g., baculovirus); plant cell systems transformedwith virus expression vectors (e.g., cauliflower mosaic virus (CaMV) andtobacco mosaic virus (TMV)), or with bacterial expression vectors (e.g.,Ti or pBR322 plasmids); or animal cell systems. The host cell employedis not limiting to the present invention.

“Control elements” or “regulatory sequences” are those non-translatedregions of the vector, e.g., enhancers, promoters, 5′ and 3′untranslated regions, which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene; LaJolla, Calif.) or PSPORT1 plasmid (Life Technologies), and the like, maybe used. The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO; and storage protein genes), or from plantviruses (e.g., viral promoters or leader sequences), may be cloned intothe vector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferred. If it is necessary to generate acell line that contains multiple copies of the sequence encodinghSLAP-2, vectors based on SV40 or EBV may be used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selected,depending upon the use intended for the expressed hSLAP-2 product. Forexample, when large quantities of expressed protein are needed for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the hSLAP-2 polypeptide, or a peptide thereof, may beligated into the vector in-frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase, so that a hybridprotein is produced; pIN vectors (See, G. Van Heeke and S. M. Schuster,1989, J. Biol. Chem., 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides, as fusion proteins with glutathione S-transferase (GST).In addition, hSLAP-2 fusion proteins expressing a His tag may begenerated, for example, in which SH2/SH3 domains from human hSLAP-2 cDNAare cloned into an expression vector linked to a poly-His tag (His).

In general, fusion proteins are soluble and can be easily purified fromlysed cells. For GST-fusion proteins purification is performed byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used (for reviews, see F. M. Ausubel et al.,supra, and Grant et al., 1987, Methods Enzymol., 153:516-544).

Should plant expression vectors be desired and used, the expression ofsequences encoding the hSLAP-2 polypeptide may be driven by any of anumber of promoters. For example, viral promoters such as the 35S and19S promoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (N. Takamatsu, 1987, EMBO J., 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO, orheat shock promoters, may be used (G. Coruzzi et al., 1984, EMBO J.,3:1671-1680; R. Broglie et al., 1984, Science, 224:838-843; and J.Winter et al., 1991, Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,S. Hobbs or L. E. Murry, In: McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express the hSLAP-2 polypeptide.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the hSLAP-2 polypeptide may be cloned into a non-essentialregion of the virus such as the polyhedrin gene and placed under controlof the polyhedrin promoter. Successful insertion of the hSLAP-2polypeptide will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the hSLAP-2 polypeptide product may be expressed (E. K.Engelhard et al., 1994, Proc. Nat. Acad. Sci., 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding the hSLAP-2 polypeptide may be ligated intoan adenovirus transcription/translation complex containing the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing the hSLAP-2 polypeptide in infected hostcells (J. Logan and T. Shenk, 1984, Proc. Natl. Acad. Sci.,81:3655-3659). In addition, transcription enhancers, such as the Roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding the hSLAP-2 polypeptide. Such signalsinclude the ATG initiation codon and adjacent sequences. In cases wheresequences encoding the hSLAP-2 polypeptide, its initiation codon, andupstream sequences are inserted into the appropriate expression vector,no additional transcriptional or translational control signals may beneeded. However, in cases where only coding sequence, or a fragmentthereof, is inserted, exogenous translational control signals, includingthe ATG initiation codon, should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system that isused, such as those described in the literature (D. Scharf et al., 1994,Results Probl. Cell Differ., 20:125-162).

Moreover, a host cell strain may be chosen for its ability to modulatethe expression of the inserted sequences or to process the expressedprotein in the desired fashion. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells having specific cellular machinery andcharacteristic mechanisms for such post-translational activities (e.g.,COS, CHO, HeLa, MDCK, HEK293, and W138) are available from the AmericanType Culture Collection (ATCC), American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209, and may be chosento ensure the correct modification and processing of the foreignprotein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe hSLAP-2 protein may be transformed using expression vectors whichmay contain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same, or on a separate,vector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched cell culture medium before they areswitched to selective medium. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows the growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the Herpes Simplex Virusthymidine kinase (HSV TK), (M. Wigler et al., 1977, Cell, 11:223-32) andadenine phosphoribosyltransferase (I. Lowy et al., 1980, Cell,22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, anti-metabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr, which confersresistance to methotrexate (M. Wigler et al., 1980, Proc. Natl. Acad.Sci., 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (F. Colbere-Garapin et al., 1981, J. Mol. Biol.,150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (S. C.Hartman and R. C. Mulligan, 1988, Proc. Natl. Acad. Sci., 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as the anthocyanins, β-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, which are widely used not onlyto identify transformants, but also to quantify the amount of transientor stable protein expression that is attributable to a specific vectorsystem (C. A. Rhodes et al., 1995, Methods Mol. Biol., 55:121-131).

Although the presence or absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thedesired gene of interest may need to be confirmed. For example, if thehSLAP-2 nucleic acid sequence polypeptide is inserted within a markergene sequence, recombinant cells containing sequences encoding thehSLAP-2 polypeptide can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding the hSLAP-2 polypeptide under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates co-expression of the tandem gene.

Alternatively, host cells which contain the nucleic acid sequenceencoding the hSLAP-2 polypeptide and which express the hSLAP-2polypeptide product may be identified by a variety of procedures knownto those having skill in the art. These procedures include, but are notlimited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques, including membrane, solution, or chip basedtechnologies, for the detection and/or quantification of nucleic acid orprotein.

Preferably, the hSLAP-2 polypeptide is substantially purified subsequentto expression. hSLAP-2 proteins can be isolated or purified in a varietyof ways known to and practiced by those having skill in the art,depending on what other components may be present in the sample.Standard purification methods include electrophoretic, molecular,immunological and chromatographic techniques, including, but not limitedto, ion exchange, hydrophobic affinity and reverse phase HPLCchromatography, and chromatofocusing. For example, the hSLAP-2 proteincan be purified using a standard antibody against hSLAP-2 column.Ultrafiltration and diafiltration techniques, in conjunction withprotein concentration, are also useful. For general guidance in suitablepurification techniques, see R. Scopes, 1982, Protein Purification,Springer-Verlag, N.Y. As will be understood by the skilled practitioner,the degree of purification necessary will vary depending on the intendeduse of the hSLAP-2 protein; in some instances, no purification will benecessary.

In addition to recombinant production, fragments of the hSLAP-2polypeptide may be produced by direct peptide synthesis usingsolid-phase techniques (J. Merrifield, 1963, J. Am. Chem. Soc.,85:2149-2154). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using ABI 431A Peptide Synthesizer (PE Biosystems). Variousfragments of the hSLAP-2 polypeptide can be chemically synthesizedseparately and then combined using chemical methods to produce thefull-length molecule.

Detection of Human hSLAP-2 Polynucleotide

The presence of polynucleotide sequences encoding the hSLAP-2polypeptide can be detected by DNA-DNA or DNA-RNA hybridization, or byamplification using probes or portions or fragments of polynucleotidesencoding the hSLAP-2 polypeptide. Nucleic acid amplification basedassays involve the use of oligonucleotides or oligomers, based on thesequences encoding the hSLAP-2 polypeptide, to detect transformantscontaining DNA or RNA encoding the hSLAP-2 polypeptide.

A wide variety of labels and conjugation techniques are known andemployed by those skilled in the art and may be used in various nucleicacid and amino acid assays. Means for producing labeled hybridization orPCR probes for detecting sequences related to polynucleotides encodingthe hSLAP-2 polypeptide include oligo-labeling, nick translation,end-labeling, or PCR amplification using a labeled nucleotide.Alternatively, the sequences encoding the hSLAP-2 polypeptide, or anyportions or fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase, such as T7, T3, orSP(6) and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (e.g., Amersham PharmaciaBiotech, Promega and U.S. Biochemical Corp.). Suitable reportermolecules or labels which may be used include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

In another of its aspects, this invention relates to a diagnostic kitfor detecting hSLAP-2 polynucleotide or polypeptide as it relates to adisease or susceptibility to a disease, particularly autoimmune diseaseswhich may be caused by hyperactivated B cells, as well as diseases whichmay be caused by hyperactivated T cells (e.g., rheumatoid arthritis;asthma; psoriasis; multiple sclerosis; rejection of organ or tissuetransplants; chronic obstructive pulmonary disease; inflammatory boweldiseases, including Crohn's Disease and ulcerative colitis; acuterespiratory distress syndrome; and systemic lupus erythematosus), ordisorders associated with other types of hematopoietic cells, such asallergies involving mast cells, leukemias and lymphomas, or chronicobstructive pulmonary disorders (as supra). Such a kit comprises one ormore of the following:

(a) a hSLAP-2 polynucleotide, preferably the nucleotide sequence of SEQID NO:1, or a fragment thereof; or

(b) a nucleotide sequence complementary to that of (a); or

(c) a hSLAP-2 polypeptide, preferably the polypeptide of SEQ ID NO: 2,or a fragment thereof; or

(d) an antibody to a hSLAP-2 polypeptide, preferably to the polypeptideof SEQ ID NO: 2, or an antibody bindable portion thereof. It will beappreciated that in any such kit, (a), (b), (c) or (d) may comprise asubstantial component and that instructions for use can be included.

Human hSLAP-2 Polypeptides—Production, Detection, Isolation

Host cells transformed with nucleotide sequences encoding the hSLAP-2protein, or fragments thereof, may be cultured under conditions suitablefor the expression and recovery of the protein from cell culture. Theprotein produced by a recombinant cell may be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those having skill in the art, expression vectorscontaining polynucleotides which encode the hSLAP-2 protein may bedesigned to contain signal sequences which direct secretion of thehSLAP-2 protein through a prokaryotic or eukaryotic cell membrane.

Other constructions may be used to join nucleic acid sequences encodingthe hSLAP-2 protein to nucleotide sequence encoding a polypeptide domainwhich will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals; protein A domains that allowpurification on immobilized immunoglobulin; and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.;Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and the hSLAP-2 protein may beused to facilitate purification.

One such expression vector provides for expression of a fusion proteincontaining hSLAP-2-encoding sequence and a nucleic acid encoding 6histidine residues preceding a thioredoxin or an enterokinase cleavagesite. The histidine residues facilitate purification on IMAC(immobilized metal ion affinity chromatography) as described by J.Porath et al., 1992, Prot. Exp. Purif., 3:263-281, while theenterokinase cleavage site provides a means for purifying from thefusion protein. For a discussion of suitable vectors for fusion proteinproduction, see D. J. Kroll et al., 1993; DNA Cell Biol., 12:441-453.

Human artificial chromosomes (HACs) may be used to deliver largerfragments of DNA than can be contained and expressed in a plasmidvector. HACs are linear microchromosomes which may contain DNA sequencesof 10 K to 10 M in size, and contain all of the elements that arerequired for stable mitotic chromosome segregation and maintenance (see,J. J. Harrington et al., 1997, Nature Genet., 15:345-355). HACs of 6 to10 M are constructed and delivered via conventional delivery methods(e.g., liposomes, polycationic amino polymers, or vesicles) fortherapeutic purposes.

A variety of protocols for detecting and measuring the expression of thehSLAP-2 polypeptide using either polyclonal or monoclonal antibodiesspecific for the protein are known and practiced in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactivewith two non-interfering epitopes on the hSLAP-2 polypeptide ispreferred, but a competitive binding assay may also be employed. Theseand other assays are described in the art as represented by thepublication of R. Hampton et al., 1990; Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn. and D. E. Maddox et al.,1983; J. Exp. Med., 158:1211-1216).

Antibodies Raised Against Human SLAP-2 and Uses Thereof

Antagonists or inhibitors of the hSLAP-2 polypeptide of the presentinvention may be produced using methods which are generally known in theart. In particular, purified hSLAP-2 protein, or fragments thereof, canbe used to produce antibodies, or to screen libraries of pharmaceuticalagents or other compounds, particularly, small molecules, synthetic ornaturally occurring, to identify those which specifically bind hSLAP-2.(e.g. Libraries are commercially available from Sigma or Aldrich).

Antibodies specific for the hSLAP-2 polypeptide, or immunogenic peptidefragments thereof, can be generated using methods that have long beenknown and conventionally practiced in the art. Such antibodies mayinclude, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab fragments, and fragments produced by an Fab expressionlibrary. Neutralizing antibodies, (i.e., those which inhibit dimerformation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, sheep, rats, mice, humans, and others, can be immunized byinjection with hSLAP-2 polypeptide, or any peptide fragment oroligopeptide thereof, which has immunogenic properties. Depending on thehost species, various adjuvants may be used to increase theimmunological response. Nonlimiting examples of suitable adjuvantsinclude Freund's (incomplete), mineral gels such as aluminum hydroxideor silica, and surface-active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Adjuvants typically used in humans include BCG (bacilli Calmette Guérin)and Corynebacterium parvumn.

Preferably, the peptides, fragments, or oligopeptides used to induceantibodies to hSLAP-2 polypeptide (i.e., immunogens) have an amino acidsequence having at least five amino acids, and more preferably, at least7-10 amino acids. It is also preferable that the immunogens areidentical to a portion of the amino acid sequence of the naturalprotein; they may also contain the entire amino acid sequence of asmall, naturally occurring molecule. The peptides, fragments oroligopeptides may comprise a single epitope or antigenic determinant ormultiple epitopes. Short stretches of hSLAP-2 amino acids may be fusedwith those of another protein, such as KLH, and antibodies are producedagainst the chimeric molecule.

Monoclonal antibodies to hSLAP-2 polypeptide, or immunogenic fragmentsthereof, may be prepared using any technique which provides for theproduction of antibody molecules by continuous cell lines in culture.These include, but are not limited to, the hybridoma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (G.Kohler et al., 1975, Nature, 256:495-497; D. Kozbor et al., 1985, J.Immunol. Methods, 81:31-42; R. J. Cote et al., 1983, Proc. Natl. Acad.Sci. USA, 80:2026-2030; and S. P. Cole et al., 1984, Mol. Cell Biol.,62:109-120). The production of monoclonal antibodies is well known androutinely used in the art.

According to the present invention, antibodies can be generated fromvarious regions of the hSLAP-2 polypeptide. Discrete domains of thehSLAP-2 protein (e.g., the proline-rich domain, or a portion thereof,the residues of which are depicted in FIGS. 3A-3B and the SH2 and/or SH3domain, or a portion thereof, the residues of which are also depicted inFIGS. 3A-3B), may also be suitable for use as immunogens to produceantibodies to human hSLAP-2.

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (S. L. Morrison et al., 1984, Proc.Natl. Acad. Sci. USA, 81:6851-6855; M. S. Neuberger et al., 1984,Nature, 312:604-608; and S. Takeda et al., 1985, Nature, 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producehSLAP-2 polypeptide-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries (D. R. Burton, 1991, Proc. Natl. Acad. Sci. USA, 88:11120-3).Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (R. Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA,86:3833-3837 and G. Winter et al., 1991, Nature, 349:293-299).

Antibody fragments which contain specific binding sites for the hSLAP-2polypeptide may also be generated. For example, such fragments include,but are not limited to, F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (W. D. Huse et al., 1989, Science, 254.1275-1281).

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve measuring the formation ofcomplexes between the hSLAP-2 polypeptide and its specific antibody. Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive with two non-interfering hSLAP-2 polypeptide epitopes ispreferred, but a competitive binding assay may also be employed (Maddox,supra).

Therapeutics/Treatments

In an embodiment of the present invention, the polynucleotide encodingthe hSLAP-2 polypeptide, or any fragment or complement thereof, may beused for therapeutic purposes. In one aspect, anti-sense to thepolynucleotide encoding the hSLAP-2 polypeptide may be used insituations in which it would be desirable to block translation of themRNA. In particular, cells may be transformed with sequencescomplementary to polynucleotides encoding the hSLAP-2 polypeptide. Thus,complementary molecules may be used to modulate human hSLAP-2polynucleotide and polypeptide activity, or to achieve regulation ofgene function. Such technology is now well known in the art, and senseor anti-sense oligomers or oligonucleotides, or larger fragments, can bedesigned from various locations along the coding or control regions ofpolynucleotide sequences encoding the hSLAP-2 polypeptide.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express nucleic acidsequence that is complementary to the nucleic acid sequence encoding thehSLAP-2 polypeptide. These techniques are described both in J. Sambrooket al., supra and in F. M. Ausubel et al., supra.

The gene encoding the hSLAP-2 polypeptide can be turned off bytransforming a cell or tissue with an expression vector that expresseshigh levels of a hSLAP-2 polypeptide-encoding polynucleotide, or afragment thereof. Such constructs may be used to introduceuntranslatable sense or anti-sense sequences into a cell. Even in theabsence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and even longer if appropriate replicationelements are designed to be part of the vector system.

Modifications of gene expression can be obtained by designing anti-sensemolecules or complementary nucleic acid sequences (DNA, RNA, or PNA), tothe control, 5′, or regulatory regions of the gene encoding the hSLAP-2polypeptide, (e.g., signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described (see, forexample, J. E. Gee et al., 1994, In: B. E. Huber and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co.; Mt. Kisco,N.Y.). The anti-sense molecule or complementary sequence may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, i.e., enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Suitableexamples include engineered hammerhead motif ribozyme molecules that canspecifically and efficiently catalyze endonucleolytic cleavage ofsequences encoding the hSLAP-2 polypeptide.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes according to theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. Such methods include techniques forchemically synthesizing oligonucleotides, for example, solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding human hSLAP-2. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP. Alternatively, the cDNA constructs that constitutively orinducibly synthesize complementary hSLAP-2 RNA can be introduced intocell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl, rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytosine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand are equally suitable for use in vivo, in vitro, and ex vivo. For exvivo therapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

The SH2 domain of SLAP was shown to bind to phosphorylated tyrosineresidues in ZAP-70, Syk, and LAT (Tang, J. et al. (1999) Proc. Natl.Acad. Sci., USA. 96: 9775-9780), and possibly other signaling proteins(Sosinowski, T. et al. (2000) J. Exptl. Med. 191: 463-474). The SH3domain of SLAP was determined to most likely bind proline rich (PR)motifs, which may help to transmit important intracellular signals inmany cell types. Seven tyrosine residues in the coding sequence ofhSLAP-2 may be sites of phosphorylation by (a) tyrosine kinase(s). Suchphosphorylated tyrosine residues may be important for binding to otherSH2- or PTB domains involved in cell regulation.

In another embodiment of the present invention, an expression vectorcontaining the complement of the polynucleotide encoding the hSLAP-2polypeptide or an anti-sense oligonucleotide, may be administered to anindividual to treat or prevent immune system related conditions,diseases, or disorders, T-cell and B-cell neoplasms; inflammationdisorders, diseases and conditions, rheumatoid arthritis,osteoarthritis, psoriasis, rhinitis, inflammatory bowel disease (Crohn'sand ulcerative colitis), allergies, particularly those involvinghyperactivity of B-cells and T-cells, or other immune cells, such asmast cells or eosinophils; autoimmune diseases such as systemic lupuserythematosus and multiple sclerosis; pulmonary diseases includingasthma, acute respiratory distress syndrome, and chronic obstructivepulmonary disorder; tissue/organ rejection; and cancer.

A variety of specialized oligonucleotide delivery techniques may beemployed, for example, encapsulation in unilamellar liposomes andreconstituted Sendai virus envelopes for RNA and DNA delivery (Arad etal., 1986, Biochem. Biophys. Acta., 859:88-94).

In another embodiment, the proteins, antagonists, antibodies,intracellular antibodies, agonists, complementary sequences, or vectorsof the present invention can be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

Any of the therapeutic methods described above may be applied to anyindividual in need of such therapy, including, for example, mammals suchas dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

Screening Methods

The hSLAP-2 protein and nucleic acid can be used in screening assays ofcandidate bioactive agents that modulate hSLAP-2 bioactivity, forpotential use to treat T- and B-cell disorders, such as tumors,lymphomas, and leukemias, or to treat inflammation disorders, such asthose involving T-cells. In addition, hSLAP-2 protein and encodingnucleic acid can be used as effectors in methods to affect T-cellactivation. By “modulate” herein is meant that the bioactivity ofhSLAP-2 is altered, i.e., either increased or decreased. In a preferredembodiment, hSLAP-2 bioactivity is inhibited. hSLAP-2 is a member of theclass of adapter proteins involved in T-cell activation and T-cellresponses; thus, it may play a role in antigen-presenting cells such asB-cells. Accordingly, hSLAP-2 can be used as a target to screen forinhibitors of its function or expression.

Inhibitors of human hSLAP-2 may be identified by screening compounds toascertain their effect on hSLAP-2 activity. As described herein, in someembodiments of the present invention, compounds are screened to identifyinhibitors by contacting human hSLAP-2 with a molecule with which itbinds or associates, (e.g., possibly ZAP-70, Syk, and LAT as suggestedby published data with the SLAP protein; Tang, J. et al. (1999) Proc.Natl. Acad Sci. USA 96:9775-9780), in the presence or absence of a testcompound. Under conditions of the assay, the inhibitors will prevent orreduce binding of human hSLAP-2 to ZAP-70, for example. Antibodies whichinhibit hSLAP-2/ZAP-70 binding are useful as inhibitors and, thereforeas positive controls in the assay.

In a similar fashion, activators of human SLAP-2 may be identified byscreening compounds to ascertain their effect on hSLAP-2/ZAP-70 binding,for example. In some embodiments of the present invention, compounds arescreened to identify activators by contacting human SLAP-2 with ZAP-70in the presence or absence of a test compound. Under conditions of theassay, the activators will enhance, accelerate or increase binding ofhuman hSLAP-2 to ZAP-70. Antibodies which inhibit hSLAP-2/ZAP-70 bindingare useful as negative controls in such assays.

In another embodiment, an assay is provided to identify compounds thatinhibit the phosphorylation of hSLAP-2 by tyrosine kinases such as, forexample but not limited to, certain cellular receptors. In one aspect,hSLAP-2 is bound to solid substrate and the reaction buffer contains³²P-gamma-ATP. Tyrosine kinase is added in the presence or absence of atest compound. Test compounds are identified that result in a decreasein the amount of ³²P label that is incorporated into hSLAP-2, comparedwith the level of phosphorylation observed in their absence. Kits areprovided which comprise a container with hSLAP-2 fixed to a solid phase,a container with the reaction buffer, optionally containing³²P-gamma-ATP, and a container with tyrosine kinase. Kits may optionallyhave positive and/or negative controls. Such kits typically also haveinstructions for performing such assays.

In another embodiment of the present invention, hSLAP-2 proteins andnucleic acids are used in screening assays to identify and detectcandidate bioactive agents that modulate hSLAP-2 bioactivity, forpotential use to treat autoimmune diseases which may be caused byhyperactivated B cells, as well as to treat diseases which may be causedby hyperactivated T cells, in addition to other immune system relatedconditions, diseases, or disorders, T-cell and B-cell neoplasms;inflammation disorders, diseases and conditions, rheumatoid arthritis,osteoarthritis, psoriasis, rhinitis, inflammatory bowel disease (Crohn'sand ulcerative colitis), allergies, particularly those involvinghyperactivity of B-cells and T-cells, or other immune cells, such asmast cells or eosinophils; autoimmune diseases such as systemic lupuserythematosus and multiple sclerosis; pulmonary diseases includingasthma, acute respiratory distress syndrome, and chronic obstructivepulmonary disorder; tissue/organ rejection; and cancer.

In a related embodiment, the methods comprise screening for a bioactiveagent capable of inhibiting the bioactivity of a hSLAP-2 protein. By“bioactivity” herein is meant the binding of the hSLAP-2 to any of itstargets, for example, including ZAP-70, Syk, and LAT, as suggested bypublished data with SLAP protein. Thus, bioactive agents that preventhSLAP-2 binding, i.e., interrupt or block or inhibit the interaction ofhSLAP-2 and its target molecule, may be found. The method comprisescombining the hSLAP-2 protein and a candidate bioactive agent, anddetermining the binding of the candidate agent to hSLAP-2 protein.

Generally, in performing such methods, a hSLAP-2 polypeptide isnon-diffusably bound to an insoluble support having isolated samplereceiving areas (e.g. a microtiter plate, an array, etc.). The criteriafor suitable insoluble supports are that they can be made of anycomposition to which polypeptides can be bound, they are readilyseparated from soluble material, and they are otherwise compatible withthe overall method of screening. The surface of such supports may besolid or porous and of any convenient size or shape. Examples ofsuitable insoluble supports include microtiter plates, arrays, membranesand beads. These are typically made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon or nitrocellulose. Microtiterplates and arrays are especially convenient, because a large number ofassays can be carried out simultaneously, using small amounts ofreagents and samples. The particular manner of binding the polypeptideis not crucial, so long as it is compatible with the reagents andoverall methods of the invention, maintains the activity of the peptideand is non-diffusable. Preferred methods of binding include the use ofantibodies (which should not hinder the binding of hSLAP-2 to itsassociated proteins), direct binding to “sticky” or ionic supports,chemical crosslinking, etc. Following binding of the polypeptide, excessunbound material is removed by washing. The sample receiving areas maythen be blocked as needed through incubation with bovine serum albumin(BSA), casein or other innocuous/non-reactive protein.

A candidate bioactive agent is added to the assay. Novel binding agentsinclude specific antibodies, non-natural binding agents identified inscreens of chemical libraries, peptide analogs, etc. Of particularinterest are screening assays for agents that have a low toxicity forhuman cells. A wide variety of assays may be used for this purpose,including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,and the like. The term “agent” as used herein describes any molecule,e.g., protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., having the capability of directly or indirectlyaltering the bioactivity of hSLAP-2 proteins. Generally a plurality ofassay mixtures are run in parallel with different agent concentrationsto obtain a differential response to the various concentrations.Typically, one of these concentrations serves as a negative control,i.e., at zero concentration, or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 10,000 daltons,preferably less than about 2000 to 5000 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. In addition, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

The determination of the binding of the candidate bioactive agent to thehSLAP-2 polypeptide may be accomplished in a number of ways practiced inthe art. In one aspect, the candidate bioactive agent is labeled, andbinding is determined directly. Where the screening assay is a bindingassay, one or more of the molecules may be joined to a label, where thelabel can directly or indirectly provide a detectable signal. Variouslabels include radioisotopes, fluorescent and chemiluminescentcompounds, specific binding molecules, particles, e.g. magneticparticles, and the like. Specific binding molecules include pairs, suchas biotin and streptavidin, digoxin and antidigoxin etc. For thespecific-binding members, the complementary member would normally belabeled with a molecule which allows detection, in accordance with knownprocedures. In some embodiments, only one of the components is labeled.Alternatively, more than one component may be labeled with differentlabels; for example, the hSLAP-2 polypeptide may be labeled with onefluorophor and the candidate agent labeled with another

In one embodiment, the candidate bioactive agent is labeled. Labeledcandidate bioactive agents are incubated with the hSLAP-2 polypeptidefor a time sufficient to allow binding, if present. Incubations may beperformed at any temperature which facilitates optimal activity,typically between 4° C. and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapid highthroughput screening. Typically between 0.1 and 1 hour is sufficient.Excess reagent is generally removed or washed away. The presence orabsence of the labeled component is detected to determine and indicatebinding.

In a preferred embodiment, the screening method comprises combining ahSLAP-2 protein, a candidate bioactive agent, and either ZAP-70 oranother of the signaling proteins that associate with hSLAP-2 (e.g.,Syk, LAT), and determining the binding of hSLAP-2 to either ZAP-70 orother signaling protein to determine the effect of the candidatebioactive agent on the hSLAP-2-signaling protein interaction.

Another embodiment of this invention encompasses small molecule (e.g.,drug) or compound screening and detection assays which involve thedetection or identification of small molecules or compounds that canbind to a given protein, i.e., the hSLAP-2 protein. Particularlypreferred are assays suitable for high throughput screeningmethodologies. In such binding-based screening or detection assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) to bescreened or assayed for binding to the protein target. Preferably, mostsmall molecules that bind to the target protein will modulate activityin some manner, due to preferential, higher affinity binding tofunctional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, hSLAP-2 polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

In a differential screening method to identity bioactive agents that arecapable of modulating the bioactivity of the hSLAP-2 protein, hSLAP-2polypeptide is combined with either ZAP-70 or another signaling moleculewhich interacts with hSLAP-2 in a first sample. A second samplecomprises a candidate bioactive agent, hSLAP-2 polypeptide and eitherZAP-70 or other hSLAP-2 interacting signaling molecule. The binding ofhSLAP-2 to either ZAP-70 or other signaling molecule is determined forboth samples, and a change, or difference in binding, between the twosamples indicates the presence of an agent capable of modulating thebioactivity of hSLAP-2. Alternatively, a differential screening methodis utilized to identify drug candidates that bind to the native hSLAP-2,but cannot bind to modified hSLAP-2 proteins, or variant hSLAP-2proteins, for example, those that have modifications which eliminate ordecrease bioactivity of a hSLAP-2 protein.

Preferably in such methods, all control and test samples are performedin at least triplicate to obtain statistically significant results.Incubation of all samples is for a time sufficient for the binding ofthe hSLAP-2 proteins and the ZAP-70 and/or other signaling protein.Following incubation, all samples are washed free of non-specificallybound material and the amount of bound, labeled material determined. Forexample, where a radiolabel is employed as a label, the samples may becounted in a scintillation counter to determine the amount of labeledcompound.

A variety of other reagents may be included in the screening assay. Suchreagents include, but are not limited to, salts, neutral proteins, e.g.albumin, detergents, etc., which may be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. In addition, reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc. may be used. Further, themixture of components in the method may be added in any order thatprovides for the requisite binding.

Kits are included as an embodiment of the present invention whichcomprise containers with reagents necessary to screen test compounds.Such kits include human hSLAP-2 and instructions for performing theassay. For example, kits may include means to detect and/or measurehuman hSLAP-2 binding using antibodies that bind to human hSLAP-2/ZAP-70complex, but not to uncomplexed proteins, or antibodies that bind touncomplexed proteins but not the human hSLAP-2/ZAP-70 complex.Optionally antibodies raised against human hSLAP-2 are provided as acontrol.

Pharmaceutical Compositions

A further embodiment of the present invention embraces theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, diluent, or excipient, for any ofthe above-described therapeutic uses and effects. Such pharmaceuticalcompositions may comprise hSLAP-2 nucleic acid, polypeptide, orpeptides, antibodies to hSLAP-2 polypeptide, or fragments thereof,mimetics, agonists (e.g., activators), antagonists (e.g., inhibitors) ofthe hSLAP-2 polypeptide or polynucleotide. The compositions may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, hormones, or biological response modifiers.

The pharmaceutical compositions for use in the present invention can beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, orrectal means.

In addition to the active ingredients (i.e., the hSLAP-2 nucleic acid orpolypeptide, or functional fragments thereof), the pharmaceuticalcompositions may contain suitable pharmaceutically acceptable carriersor excipients comprising auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration areprovided in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained by thecombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropyl-methylcellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth, andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a physiologically acceptable saltthereof, such as sodium alginate.

Dragee cores may be used in conjunction with physiologically suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification, or tocharacterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations, which can be used orally, include push-fitcapsules made of gelatin, as well as soft, scaled capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. In addition,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyloleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants or permeation agentsthat are appropriate to the particular barrier to be permeated are usedin the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Saltstend to be more soluble in aqueous solvents, or other protonic solvents,than are the corresponding free base forms. In other cases, thepreferred preparation may be a lyophilized powder which may contain anyor all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7%mannitol, at a pH range of 4.5 to 5.5, combined with a buffer prior touse. After the pharmaceutical compositions have been prepared, they canbe placed in an appropriate container and labeled for treatment of anindicated condition. For administration of the hSLAP-2 product, suchlabeling would include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose or amount is well within the capability of thoseskilled in the art. For any compound, the therapeutically effective dosecan be estimated initially either in cell culture assays, e.g., usingneoplastic cells, or in animal models, usually mice, rabbits, dogs, orpigs. The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used and extrapolated to determine useful doses and routes foradministration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example, the hSLAP-2 polypeptide, or active fragmentsthereof, antibodies to the hSLAP-2 polypeptide, agonists or antagonistsof the hSLAP-2 polypeptide, which ameliorates, reduces, or eliminatesthe symptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the ratio, LD₅₀/ED₅₀.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in determining a range of dosages for human use. Preferreddosage contained in a pharmaceutical composition is within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, who willconsider the factors related to the individual requiring treatment.Dosage and administration are adjusted to provide sufficient levels ofthe active moiety or to maintain the desired effect. Factors which maybe taken into account include the severity of the individual's diseasestate, general health of the patient, age, weight, and gender of thepatient, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. As a general guide, long-acting pharmaceutical compositions maybe administered every 3 to 4 days, every week, or once every two weeks,depending on half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms (μg), upto a total dose of about 1 gram (g), depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and is generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, and the like.

Assays and Diagnostics

In another embodiment of the present invention, antibodies whichspecifically bind to the hSLAP-2 polypeptide may be used for thediagnosis of conditions or diseases characterized by expression (oroverexpression) of the hSLAP-2 polynucleotide or polypeptide, or inassays to monitor patients being treated with hSLAP-2 polypeptide, orits agonists, antagonists, or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for use in therapeutic methods. Diagnostic assays forthe hSLAP-2 polypeptide include methods which utilize the antibody and alabel to detect the protein in human body fluids or extracts of cells ortissues. The antibodies may be used with or without modification, andmay be labeled by joining them, either covalently or non-covalently,with a reporter molecule. A wide variety of reporter molecules which areknown in the art may be used, several of which are described above.

Several assay protocols including ELISA, RIA, and FACS for measuring thehSLAP-2 polypeptide are known in the art and provide a basis fordiagnosing altered or abnormal levels of hSLAP-2 polypeptide expression.Normal or standard values for hSLAP-2 polypeptide expression areestablished by combining body fluids or cell extracts taken from normalmammalian subjects, preferably human, with antibody to the hSLAP-2polypeptide under conditions suitable for complex formation. The amountof standard complex formation may be quantified by various methods;photometric means are preferred. Quantities of the hSLAP-2 polypeptideexpressed in subject sample, control sample, and disease samples frombiopsied tissues are compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease.

According to another embodiment of the present invention, thepolynucleotides encoding hSLAP-2 polypeptide may be used for diagnosticpurposes. The polynucleotides which may be used include oligonucleotidesequences, complementary RNA and DNA molecules, and PNAs. Thepolynucleotides may be used to detect and quantify hSLAP-2-encodingnucleic acid expression in biopsied tissues in which expression (orunder- or over-expression) of hSLAP-2 polynucleotide may be correlatedwith disease. The diagnostic assay may be used to distinguish betweenthe absence, presence, and excess expression of hSLAP-2, and to monitorregulation of hSLAP-2 polynucleotide levels during therapeutic treatmentor intervention.

In a related aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding hSLAP-2 polypeptide, or closely related molecules, may be usedto identify nucleic acid sequences which encode the hSLAP-2 polypeptide.The specificity of the probe, whether it is made from a highly specificregion, e.g., about 8 to 10 or 12 or 15 contiguous nucleotides in the 5′regulatory region, or a less specific region, e.g., especially in the 3′coding region, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low) will determine whether the probeidentifies only naturally occurring sequences encoding the hSLAP-2polypeptide, alleles thereof, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50%, preferably greater than 80%, ofthe nucleotides encoding hSLAP-2 polypeptide. The hybridization probesof this invention may be DNA or RNA and may be derived from thenucleotide sequence of SEQ ID NO:1, or from genomic sequence includingpromoter, enhancer elements, and introns of the naturally occurringhSLAP-2 protein.

Methods for producing specific hybridization probes for DNA encoding thehSLAP-2 polypeptide include the cloning of nucleic acid sequence thatencodes the hSLAP-2 polypeptide, or hSLAP-2 derivatives, into vectorsfor the production of mRNA probes. Such vectors are known in the art,commercially available, and may be used to synthesize RNA probes invitro by means of the addition of the appropriate RNA polymerases andthe appropriate labeled nucleotides. Hybridization probes may be labeledby a variety of detector/reporter groups, e.g., radionuclides such as³²P or ³⁵S, or enzymatic labels, such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems, and the like.

The polynucleotide sequence encoding the hSLAP-2 polypeptide may be usedin Southern or Northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detect thestatus of, e.g., levels or overexpression of hSLAP-2, or to detectaltered hSLAP-2 expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequence encoding the hSLAP-2polypeptide may be useful in assays that detect activation or inductionof various B- and T-cell-related neoplasms or cancers, particularlythose mentioned supra. The nucleotide sequence encoding the hSLAP-2polypeptide may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantified and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequence has hybridized with nucleotide sequencepresent in the sample, and the presence of altered levels of nucleotidesequence encoding the hSLAP-2 polypeptide in the sample indicates thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

To provide a basis for the diagnosis of disease associated withexpression of hSLAP-2, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes the hSLAP-2 polypeptide,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject (patient) values is used to establish the presenceof disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in a normal individual. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcriptin biopsied tissue from an individual may indicate a predisposition forthe development of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier, therebypreventing the development or further progression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thenucleic acid sequence encoding the hSLAP-2 polypeptide may involve theuse of PCR. Such oligomers may be chemically synthesized, generatedenzymatically, or produced from a recombinant source. Oligomers willpreferably comprise two nucleotide sequences, one with sense orientation(5′→3′) and another with anti-sense (3′→5′), employed under optimizedconditions for identification of a specific gene or condition. The sametwo oligomers, nested sets of oligomers, or even a degenerate pool ofoligomers may be employed under less stringent conditions for detectionand/or quantification of closely related DNA or RNA sequences.

Methods suitable for quantifying the expression of hSLAP-2 includeradiolabeling or biotinylating nucleotides, co-amplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (P. C. Melby et al., 1993, J. Immunol. Methods,159:235-244; and C. Duplaa et al., 1993, Anal. Biochem., 229-236). Thespeed of quantifying multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantification.

In another embodiment of the present invention, oligonucleotides, orlonger fragments derived from the hSLAP-2 polynucleotide sequencedescribed herein may be used as targets in a microarray. The microarraycan be used to monitor the expression level of large numbers of genessimultaneously (to produce a transcript image), and to identify geneticvariants, mutations and polymorphisms. This information may be used todetermine gene function, to understand the genetic basis of a disease,to diagnose disease, and to develop and monitor the activities oftherapeutic agents. In a particular aspect, the microarray is preparedand used according to the methods described in WO 95/11995 (Chee etal.); D. J. Lockhart et al., 1996, Nature Biotechnology, 14:1675-1680;and M. Schena et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-10619).Microarrays are further described in U.S. Pat. No. 6,015,702 to P. Lalet al.

In another embodiment of this invention, the nucleic acid sequence whichencodes the hSLAP-2 polypeptide may also be used to generatehybridization probes which are useful for mapping the naturallyoccurring genomic sequence. The sequences may be mapped to a particularchromosome, to a specific region of a chromosome, or to artificialchromosome constructions (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial PI constructions, orsingle chromosome cDNA libraries, as reviewed by C. M. Price, 1993,Blood Rev., 7:127-134 and by B. J. Trask, 1991, Trends Genet.,7:149-154.

In another embodiment of the present invention, the hSLAP-2 polypeptide,its catalytic or immunogenic fragments or oligopeptides thereof, can beused for screening libraries of compounds in any of a variety of drugscreening techniques. The fragment employed in such screening may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenthe hSLAP-2 polypeptide, or portion thereof, and the agent being tested,may be measured utilizing techniques commonly practiced in the art andas described above.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest, for example, as described in WO 84/03564. Inthis method, as applied to the hSLAP-2 protein, large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with the hSLAP-2 polypeptide, or fragments thereof, and washed.Bound hSLAP-2 polypeptide is then detected by methods well known in theart. Purified hSLAP-2 polypeptide can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In a further embodiment of this invention, competitive drug screeningassays can be used in which neutralizing antibodies capable of bindinghSLAP-2 polypeptide specifically compete with a test compound forbinding to hSLAP-2 polypeptide. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with the hSLAP-2 polypeptide.

Transgenics and Knock Outs

The present invention further encompasses transgenic non-human mammals,preferably mice, that comprise a recombinant expression vector harboringa nucleic acid sequence that encodes human hSLAP-2 comprising the aminoacid sequence of SEQ ID NO:2.

Transgenic non-human mammals useful to produce recombinant proteins arewell known to the skilled practitioner, as are the expression vectorsnecessary and the techniques for generating transgenic animals.Generally, the transgenic animal comprises a recombinant expressionvector in which the nucleotide sequence that encodes human hSLAP-2 isoperably linked to a tissue specific promoter whereby the codingsequence is only expressed in that specific tissue. For example, thetissue specific promoter can be a mammary cell specific promoter and therecombinant protein so expressed is recovered from the animal's milk.

The transgenic animals, particularly transgenic mice, containing anucleic acid molecule which encodes human hSLAP-2 may be used as animalmodels for studying in vivo the overexpression of hSLAP-2 and for use indrug evaluation and discovery efforts to find compounds effective toinhibit or modulate the activity of hSLAP-2, such as, for example,compounds for treating immune system related conditions, diseases, ordisorders, T-cell and B-cell neoplasms; inflammation disorders, diseasesand conditions, rheumatoid arthritis, osteoarthritis, psoriasis,rhinitis, inflammatory bowel disease (Crohn's and ulcerative colitis),allergies, particularly those involving hyperactivity of B-cells andT-cells, or other immune cells, such as mast cells or eosinophils;autoimmune diseases such as systemic lupus erythematosus and multiplesclerosis; pulmonary diseases including asthma, acute respiratorydistress syndrome, and chronic obstructive pulmonary disorder;tissue/organ rejection; and cancer. One having ordinary skill in the artusing standard techniques, such as those taught in U.S. Pat. No.4,873,191, issued Oct. 10, 1989 to Wagner et al. and in U.S. Pat. No.4,736,866, issued Apr. 12, 1988 to Leder et al., can produce transgenicanimals which produce the human hSLAP-2, or splice variants thereof, anduse the animals in drug evaluation and discovery projects.

Another aspect of the present invention relates to knockout mice andmethods of using the same. In particular, transgenic mice may begenerated which are homozygous for a mutated, non-functional hSLAP-2gene which is introduced into the animals using well-known techniques.The knockout mice produce no functional hSLAP-2 and thus are useful tostudy the function of hSLAP-2. Furthermore, the mice may be used inassays to study the effect of test compounds in hSLAP-2 deficientanimals. For instance, hSLAP-2-deficient mice can be used to determineif, how and to what extent hSLAP-2 inhibitors will effect the animal andthus address concerns associated with inhibiting the activity of themolecule.

Methods of generating genetically deficient “knockout” mice are wellknown and are disclosed in M. R. Capecchi, 1989, Science, 244:1288-1292and P. Li et al., 1995, Cell, 80:401-411. The human hSLAP-2 cDNA clonecan be used to isolate a murine hSLAP-2 genomic clone. The genomic clonecan be used to prepare a hSLAP-2 targeting construct which can disruptthe hSLAP-2 gene in the mouse by homologous recombination. The targetingconstruct contains a non-functioning portion of the hSLAP-2 gene whichinserts in place of the functioning portion of the native mouse gene.The non-functioning insert generally contains an insertion in the exonthat encodes the active region of hSLAP-2. The targeting construct cancontain markers for both positive and negative selection. The positiveselection marker allows for the selective elimination of cells which donot carry the marker, while the negative selection marker allows for theelimination of cells that carry the marker.

For example, a first selectable marker is a positive marker that willallow for the survival of cells carrying it. In some instances, thefirst selectable marker is an antibiotic resistance gene, such as theneomycin resistance gene, which can be placed within the coding sequenceof the hSLAP-2 gene to render it non-functional, while at the same timerendering the construct selectable. The antibiotic resistance gene iswithin the homologous region which can recombine with native sequences.Thus, upon homologous recombination, the non-functional and antibioticresistance selectable gene sequences will be taken up. Knockout mice maybe used as models, in particular, the Cre-Lox model, for studying B- andT-cell related disorder and hyperactivity and screening compounds fortreating these disorders.

The targeting construct also contains a second selectable marker whichis a negative selectable marker. Cells with the negative selectablemarker will be eliminated. The second selectable marker is outside therecombination region. Thus, if the entire construct is present in thecell, both markers will be present. If the construct has recombined withnative sequences, the first selectable marker will be incorporated intothe genome and the second will be lost. The herpes simplex virusthymidine kinase (HSV tk) gene is an example of a negative selectablemarker which can be used as a second marker to eliminate cells thatcarry it. Cells with the HSV tk gene are selectively killed in thepresence of gangcyclovir.

Cells are transfected with targeting constructs and then selected forthe presence of the first selection marker and the absence of thesecond. Constructs/DNA are then injected into the blastocyst stage andimplanted into pseudopregnant females. Chimeric offspring which arecapable of transferring the recombinant genes in their germline areselected, mated and their offspring examined for heterozygous carriersof the recombined genes. Mating of the heterozygous offspring can thenbe used to generate fully homozygous offspring which constitutehSLAP-2-deficient knockout mice.

EXAMPLES

The Examples below are provided to illustrate the subject invention andare not intended to limit the invention.

Example 1 Methods

Cloning of the Full Length Human hSLAP-2 Gene

The LifeSeq EST database (Incyte Pharmaceuticals, Inc., California) wasscreened for novel SH2/SH3 domain genes using an SH2/SH3 domain hiddenMarkov model (HMM) from the Pfam database (A. Bateman et al., 2000, “ThePfam protein families database”, Nucleic Acids Res., 28:263-266) and theGenewise/Wise2 software package (Wise2 Documentation (version 2.1.20stable), Ewan Birney, Richard Copley Sanger Centre, Wellcome TrustGenome Campus, Hinxton, Cambridge B 10 1SA, England;http://www.sanger.ac.uk/Software/Wise2/wisedocs/wise2/wise2.html). Onenovel SH2/SH3 domain-containing sequence was identified (clone 3182427).Sequencing and analysis of this clone indicated that it contained thefull length coding region of a new gene.

To further elucidate the complete structure of this gene, full-lengthcloning experiments were performed using the GeneTrapper™(LifeTechnologies, Inc.; Gaithersburg, Md.). Briefly, PCR primers PY749(5′-CGGATCAGACACTACAGGATC-3′), (SEQ ID NO:3) and PY751(5′-CGTCATTCAGGCTGATGTAG-3′), (SEQ ID NO:4) were used to screen thehuman leukocyte cDNA library. The GeneTrapper™ primer, PY750(5′-TACTCTGAGCTGGCGGATGACATCTGCTGC-3′), (SEQ ID NO:5), used to screen ahuman leukocyte cDNA library (Life Technologies), identified positiveclones containing the novel sequence. End-sequencing analysis revealedthat one of the clones (clone #13) contained the full-length codingregion of the new gene. The entire clone was subjected to sequenceanalysis with additional primers. The vector for this cDNA insert ispCMVSPORT2 with cloning sites SalI (5′-end) and NotI (3′-end). Sequenceanalyses were performed using the GCG/Wisconsin package (GeneticsComputer Group, Madison, Wis.).

Sequence Analysis

As shown in FIGS. 1-3, sequencing of one of the isolated cDNA clones,clone #13, showed that this isolate has a 2567 nucleotide coding region,encoding a polypeptide of 261 amino acids. Sequence analysis revealedthat the new gene has an SH3 domain (residues 35 to 90) and a SH2/SH3domain (residues 94 to 176). Database searching against human genesshowed that this novel protein exhibits the highest level of homology(˜47% identity and ˜58% similarity over 219 amino acids) with hSLAP(FIGS. 4-7). In addition, the new gene also shows significant homologyto the SH2/SH3/SH3 regions of the Lyn and Hck tyrosine kinases from theSrc-family (41% identity over 172 amino acids) which would be expectedin this SLAP-family of proteins.

Example 2 Northern Analysis

Northern analysis is used to detect the presence of a transcript of agene and involves the hybridization of a labeled nucleotide sequence toa membrane on which RNA from a particular cell or tissue type has beenbound (see, J. Sambrook et al., supra). Electronic Northern analysisindicated that the novel gene of the present invention had a restrictedexpression profile, demonstrating detection of mRNAs in immune systemtissues, embryonic structures, and the digestive tract (FIG. 8). Immunesystem cells included peripheral blood lymphocytes, Jurkat T-cells andbone-marrow cells. Embryonic structures included the placenta and thedigestive tract included both the colon and the small intestine. Theplacenta may have been positive for expression due to the presence ofimmune system cells, and intestinal tissue may have been positive forexpression due to the presence of intraepithelial lymphocytes (IELs).Analogous computer techniques using BLAST (S. F. Altschul, 1993, J. Mol.Evol., 36:290-300 and S. F. Altschul et al., 1990, J. Mol. Evol.,215:403-410) can be used to search for identical or related molecules innucleotide databases, such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much more rapid and lesslabor-intensive than performing multiple, membrane-based hybridizations.In addition, the sensitivity of the computer search can be modified todetermine whether any particular match is categorized as being exact(identical) or homologous.

The basis of the search is the product score, which is defined asfollows: (% sequence identity×maximum BLAST score)/100. The productscore takes into account both the degree of similarity between twosequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; at 70,the match will be exact. Homologous molecules are usually identified byselecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules. The results of Northernanalysis are reported as a list of libraries in which the transcriptencoding hSLAP-2 occurs. Abundance and percent abundance are alsoreported. Abundance directly reflects the number of times that aparticular transcript is represented in a cDNA library, and percentabundance is abundance divided by the total number of sequences that areexamined in the cDNA library.

Example 3 Labeling of Hybridization Probes and Use Thereof

Hybridization probes derived from SEQ ID NO:1 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotidescontaining about 20 base pairs is described in this Example, essentiallythe same procedure is used with larger cDNA fragments. Oligonucleotidesare designed using state-of-the-art software such as OLIGO 4.06(National Biosciences), labeled by combining 50 pmol of each oligomerand 250 μCi of [γ-³²P] adenosine triphosphate (Amersham) and T4polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeledoligonucleotides are substantially purified with SEPHADEX G-25 superfineresin column (Amersham Pharmacia Biotech). A portion containing 10⁷counts per minute of each of the sense and anti-sense oligonucleotidesis used in a typical membrane based hybridization analysis of humangenomic DNA digested with one of the following endonucleases (e.g., AseI, Bg1 II, Eco RI, Pst I, Xba 1, or Pvu II, DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMATAR film(Kodak; Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics; Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

Example 4 Complementary Polynucleotides

Anti-sense molecules or nucleic acid sequence complementary to thehSLAP-2 protein-encoding sequence, or any part thereof, is used todecrease or to inhibit the expression of naturally occurring hSLAP-2.Although the use of anti-sense or complementary oligonucleotidescomprising about 15 to 35 base-pairs is described, essentially the sameprocedure is used with smaller or larger nucleic acid sequencefragments. An oligonucleotide based on the coding sequence of hSLAP-2protein, as shown in FIGS. 2 and 3A-3B, is used to inhibit expression ofnaturally occurring hSLAP-2. The complementary oligonucleotide isdesigned from the most unique 5′ sequence (FIGS. 1 and 3A-3B), and isused either to inhibit transcription by preventing promoter binding tothe coding sequence, or to inhibit translation by preventing theribosome from binding to the hSLAP-2 protein-encoding transcript. Usingan appropriate portion of the signal and 5′ sequence of SEQ ID NO:1, aneffective anti-sense oligonucleotide includes any of about 15-35nucleotides spanning the region which translates into the signal or 5′coding sequence of the polypeptide as shown in FIGS. 2 and 3A-3B.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe hSLAP-2 protein coding sequence (SEQ ID NO:1).

Example 5 Microarrays

For the production of oligonucleotides for a microarray, SEQ ID NO:1 isexamined using a computer algorithm which starts at the 3 prime end ofthe nucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangethat is suitable for hybridization and lack predicted secondarystructure that would interfere with hybridization. The algorithmidentifies specific oligonucleotides of 20 nucleotides in length, i.e.,20-mers. A matched set of oligonucleotides is created in which onenucleotide in the center of each sequence is altered. This process isrepeated for each gene in the microarray, and double sets of 20-mers aresynthesized in the presence of fluorescent or radioactive nucleotidesand arranged on the surface of a substrate. When the substrate is asilicon chip, a light-directed chemical process is used for deposition(WO 95/11995, M. Chee et al.).

Alternatively, a chemical coupling procedure and an ink jet device isused to synthesize oligomers on the surface of a substrate. (WO95/25116, J. D. Baldeschweiler et al.). As another alternative, a“gridded” array that is analogous to a dot (or slot) blot is used toarrange and link cDNA fragments or oligonucleotides to the surface of asubstrate using, for example, a vacuum system, or thermal, UV,mechanical, or chemical bonding techniques. A typical array may beproduced by hand, or by using available materials and equipment, and maycontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots, or 6144dots. After hybridization, the microarray is washed to remove anynon-hybridized probe, and a detection device is used to determine thelevels and patterns of radioactivity or fluorescence. The detectiondevice may be as simple as X-ray film, or as complicated as a lightscanning apparatus. Scanned fluorescent images are examined to determinedegree of complementarity and the relative abundance/expression level ofeach oligonucleotide sequence in the microarray.

Example 6 Purification of Naturally Occurring HSLAP-2 Protein UsingSpecific Antibodies

Naturally occurring or recombinant hSLAP-2 polypeptide is substantiallypurified by immunoaffinity chromatography using antibodies specific forthe hSLAP-2 polypeptide, or a peptide derived therefrom. Animmunoaffinity column is constructed by covalently coupling polypeptideantibody raised against hSLAP-2 to an activated chromatographic resin,such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech, Inc.;Piscataway, N.J.). After the coupling, the resin is blocked and washedaccording to the manufacturer's instructions.

Medium containing hSLAP-2 polypeptide is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of the hSLAP-2 polypeptide (e.g., high ionicstrength buffers in the presence of detergent). The column is elutedunder conditions that disrupt antibody/hSLAP-2 polypeptide binding(e.g., a buffer of pH 2-3, or a high concentration of a chaotrope, suchas urea or thiocyanate ion), and hSLAP-2 polypeptide is collected.

Example 7 Identification of Molecules that Interact with the HumanSLAP-2 Protein

hSLAP-2 polypeptide, or biologically active fragments thereof, arelabeled with ¹²⁵I Bolton-Hunter reagent (Bolton et al., 1973, Biochem.J, 133:529). Candidate molecules previously arrayed in wells of amulti-welled plate are incubated with the labeled hSLAP-2 polypeptide,washed, and any wells having labeled hSLAP-2 polypeptide-candidatemolecule complexes are assayed. Data obtained using differentconcentrations of the hSLAP-2 polypeptide are used to calculate valuesfor the number, affinity and association of the hSLAP-2 polypeptide withthe candidate molecules. In addition, data may be obtained using fusionproteins such as GST- or polyhistidine tagged fusion proteins,co-immunoprecipitation and/or Western immunoblotting, etc.

The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals andabstracts cited herein are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

1-20. (canceled)
 21. An isolated polypeptide comprising a polypeptidesequence selected from the group consisting of: (a) an isolatedpolypeptide comprising amino acids 1 to 261 of SEQ ID NO:2; (b) anisolated polypeptide comprising amino acids 2 to 261 of SEQ ID NO:2; (c)an isolated polypeptide comprising a polypeptide sequence encoded bynucleotides 415 to 1197 of SEQ ID NO:1; and (d) an isolated polypeptidecomprising a polypeptide sequence encoded by nucleotides 418 to 1197 ofSEQ ID NO:1.
 22. The isolated polypeptide of claim 21, wherein saidpolypeptide is (a).
 23. The isolated polypeptide of claim 21, whereinsaid polypeptide is (b).
 24. The isolated polypeptide of claim 21,wherein said polypeptide is (c).
 25. The isolated polypeptide of claim21, wherein said polypeptide is (d).
 26. An isolated polypeptideproduced by a method comprising: (a) culturing an isolated recombinanthost cell comprising a vector that comprises the coding region encodingthe polypeptide of claim 21 under conditions such that the polypeptideof claim 21 is expressed; and (b) recovering said polypeptide.
 27. Anisolated polypeptide comprising the polypeptide encoded by the hSLAP-2cDNA clone contained in ATCC Deposit No: PTA-3873.
 28. An isolatedpolypeptide consisting of amino acids from position 35 to position 90 ofSEQ ID NO:2.
 29. An isolated polypeptide consisting of amino acids fromposition 94 to position 176 of SEQ ID NO:2.
 30. The isolated polypeptideof claim 21 wherein said polypeptide contains a single amino acidsubstitution.
 31. An isolated polypeptide having as least 95.0% identityto amino acids 2 to 261 of SEQ ID NO:2, wherein percent identity iscalculated using a CLUSTALW sequence alignment.